AU725196B2

AU725196B2 - Seed plants exhibiting inducible early reproductive development and methods of making same

Info

Publication number: AU725196B2
Application number: AU33000/97A
Authority: AU
Inventors: Martin F. Yanofsky
Original assignee: University of California
Current assignee: University of California
Priority date: 1996-06-05
Filing date: 1997-06-04
Publication date: 2000-10-05
Anticipated expiration: 2017-06-04
Also published as: AU3300097A; US6355863B1; WO1997046079A1; US6002069A

Description

WO 97/46079 PCT/US97/09682 1 SEED PLANTS EXHIBITING INDUCIBLE EARLY REPRODUCTIVE DEVELOPMENT AND METHODS OF MAKING SAME This work was supported by grant DCB-9018749 awarded by the National Science Foundation and by grant USDA 93-37304 awarded by the United States Department of Agriculture. The United States Government has certain rights in this invention.

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION This invention relates generally to the field of plant genetic engineering and more specifically to genes involved in the regulation of plant reproductive development.

BACKGROUND INFORMATION A flower is the reproductive structure of a flowering plant. Following fertilization, the ovary of the flower becomes a fruit and bears seeds. As a practical consequence, production of fruit and seed-derived crops such as grapes, beans, corn, wheat, rice and hops is dependent upon flowering.

Early in the life cycle of a flowering plant, vegetative growth occurs, and roots, stems and leaves are formed. During the later period of reproductive growth, flowers as well as new shoots or branches develop.

However, the factors responsible for the transition from vegetative to reproductive growth, and the onset of flowering, are poorly understood.

WO 97/46079 PCT/US97/09682 2 A variety of external signals, such as length of daylight and temperature, affect the time of flowering. The time of flowering also is subject to genetic controls that prevent young plants from flowering prematurely. Thus, the pattern of genes expressed in a plant is an important determinant of the time of flowering.

Given these external signals and genetic controls, a relatively fixed period of vegetative growth precedes flowering in a particular plant species. The length of time required for a crop to mature to flowering limits the geographic location in which it can be grown and can be an important determinant of yield. In addition, since the time of flowering determines when a plant is reproductively mature, the pace of a plant breeding program also depends upon the length of time required for a plant to flower.

Traditionally, plant breeding involves generating hybrids of existing plants, which are examined for improved yield or quality. The improvement of existing plant crops through plant breeding is central to increasing the amount of food grown in the world since the amount of land suitable for agriculture is limited.

For example, the development of new strains of wheat, corn and rice through plant breeding has increased the yield of these crops grown in underdeveloped countries such as Mexico, India and Pakistan. Unfortunately, plant breeding is inherently a slow process since plants must be reproductively mature before selective breeding can proceed.

For some plant species, the length of time needed to mature to flowering is so long that selective breeding, which requires several rounds of backcrossing progeny plants with their parents, is impractical. For 3 example, perennial trees such as walnut, hickory, oak, maple and cherry do not flower for several years after planting. As a result, breeding of such plant species for insect or disease-resistance or to produce improved wood or fruit, for example, would require decades, even if only a few rounds of selection were performed.

Methods of promoting early reproductive development can make breeding of long generation seed plants such as trees practical for the first time.

Methods of promoting early reproductive development also would be useful for shortening growth periods, thereby broadening the geographic range in which a crop such as rice, corn or coffee can be grown. Unfortunately, methods for promoting early reproductive development in a seed plant have not yet been described. Thus, there is a need for methods that promote early reproductive development. The present invention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION According to a first aspect of the present invention there is provided a recombinant nucleic acid molecule, comprising an inducible regulatory element operably linked to a nucleic acid molecule encoding .CAULIFLOWER (CAL). An inducible regulatory element can be, for example, a copper inducible regulatory element, 30 tetracycline inducible regulatory element, ecdysone o: inducible regulatory element or heat-shock inducible :....:regulatory element. The invention further provides a oo :transgenic seed plant, such as an angiosperm or gymnosperm, that contains a recombinant nucleic acid molecule of the invention.

PR IS. 1 1 L H 1 I 19 )1 4 According to a second aspect of the present invention there is provided a method of converting shoot meristem to floral meristem in an angiosperm, comprising the steps of: introducing into said angiosperm a recombinant nucleic acid molecule comprising an inducible regulatory element operably linked to a nucleic acid molecule encoding CAULIFLOWER (CAL) to produce a transgenic angiosperm; and contacting said transgenic angiosperm with an inducing agent, thereby increasing expression of said CAL and converting shoot meristem to floral meristem in said transgenic angiosperm.

The methods of the invention can be practiced with an inducible regulatory element such as a copper inducible regulatory element, tetracycline inducible regulatory element, ecdysone inducible regulatory element or heat-shock inducible regulatory element.

According to a third aspect of the present invention there is provided a method of promoting early reproductive development in a seed plant, comprising the steps of: :oo: introducing into said seed plant said recombinant nucleic acid molecule comprising an inducible 25 regulatory element operably linked to a nucleic acid molecule encoding CAULIFLOWER (CAL) to produce a transgenic seed plant; and contacting said transgenic seed plant with an inducing agent, thereby increasing expression of said CAL and promoting early reproductive development in said transgenic seed plant.

According to a fourth aspect of the present invention there is provided a nucleic acid molecule encoding a chimeric protein, comprising a nucleic acid molecule encoding a floral meristem identity gene product linked in frame to a nucleic acid molecule encoding a ligand binding domain. A transgenic seed plant, such as I~onme Isbel\ Speci\,32967 doc 19/07/00 an angiosperm or gymnosperm, that contains a nucleic acid molecule encoding a chimeric protein of the invention also is provided.

According to a fifth aspect of the present invention there is provided a method of converting shoot meristem to floral meristem in an angiosperm by introducing a nucleic acid molecule encoding a chimeric protein of the invention into the angiosperm to produce a transgenic angiosperm, where, under appropriate conditions, the chimeric protein containing a floral meristem identity gene product fused to a ligand binding domain is expressed; and contacting the transgenic angiosperm with cognate ligand, where, upon binding of cognate ligand to the ligand binding domain, floral meristem identity gene product activity is increased, thereby converting shoot meristem to floral meristem in the transgenic angiosperm. A floral meristem identity gene product useful in converting shoot meristem to floral meristem can be, for example, AP1, CAL or LFY, and a ligand binding domain can be, for example, a glucocorticoid receptor ligand binding domain or an ecdysone receptor ligand binding domain According to a sixth aspect of the present invention there is provided a method of 25 promoting early reproductive development in a seed plant by introducing a nucleic acid molecule encoding a chimeric protein of the invention into the seed plant to produce a transgenic seed plant, where, under appropriate conditions, the chimeric protein containing a floral meristem identity gene product fused to a ligand binding domain is expressed; and contacting the transgenic seed S: plant with cognate ligand, where, upon binding of the cognate ligand to the ligand binding domain, floral meristem identity gene product activity is increased, thereby promoting early reproductive development in the transgenic seed plant. A floral meristem identity gene k product such as API, CAL or LFY and a ligand binding BPI.. I ;1"07 'A 6 domain such as a glucocorticoid receptor ligand binding domain or an ecdysone receptor ligand binding domain are particularly useful in the methods of the invention for promoting early reproductive development.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates a western-blot analysis of tissues from wild type and mutant Arabidopsis plants with anti-API antisera.

DETAILED DESCRIPTION OF THE INVENTION In general terms, the invention relates to a nonnaturally occurring seed plant containing a first ectopically expressible nucleic acid molecule encoding a first floral meristem identity gene product, provided that the nucleic acid molecule is not ectopically expressed due to a mutation in an endogenous TERMINAL FLOWER gene. For example, the transgenic seed plant may contain a first extopically expressible floral meristem identity gene product such as APETALA1 (APl), CAULIFLOWER (CAL) or LEAFY (LFY). A transgenic seed plant can be, for example, an angiosperm such as a cereal plant, leguminous plant, oilseed plant, hardwood tree, fruit-bearing plant or ornamental flower or a gymnosperm such as a coniferous tree.

A flower, like a leaf or shoot, is derived from the shoot apical meristem, which is a collection of undifferentiated cells set aside during embryogenesis.

The production of vegetative structures, such as leaves or shoots, and of reproductive structures, such as flowers, is temporally segregated, such that a leaf or shoot arises early in a plant life cycle, while a flower develops later. The transition from vegetative to reproductive development is the consequence of a process I~P l Th~,1I1Sip~Ci296 d oc 19,07/00 6a termed floral induction (Yanof sky, Ann. Rev. Plant Physiol. Plant Mol. Biol. 46:167-188 (1995), which is incorporated herein by reference).

%"RP S I It ,12. rlcc [9,07/00 WO 97/46079 PCT/US97/09682 7 Once induced, shoot apical meristem either persists and produces floral meristem, which gives rise to flowers, and lateral meristem, which gives rise to branches, or is itself converted to floral meristem.

Floral meristem differentiates into a single flower having a fixed number of floral organs in a whorled arrangement. Dicots, for example, contain four whorls (concentric rings), in which sepals (first whorl) and petals (second whorl) surround stamens (third whorl) and carpels (fourth whorl).

Although shoot meristem and floral meristem both consist of meristemic tissue, shoot meristem is distinguishable from the more specialized floral meristem. Shoot meristem generally is indeterminate and gives rise to an unspecified number of floral and lateral meristems. In contrast, floral meristem is determinate and gives rise to the fixed number of floral organs that comprise a flower.

By convention herein, a wild-type gene sequence is represented in upper case italic letters (for example, APETALAI), and a wild-type gene product is represented in upper case non-italic letters (APETALA1). Further, a mutant gene allele is represented in lower case italic letters (apl), and a mutant gene product is represented in lower case non-italic letters (apl).

Genetic studies have identified a number of genes involved in regulating flower development. These genes can be classified into different groups depending on their function. Flowering time genes, for example, are involved in floral induction and regulate the transition from vegetative to reproductive growth. In comparison, the floral meristem identity genes, which are the subject matter of the present invention as disclosed herein, encode proteins that promote the conversion of WO 97/46079 PCT/US97/09682 8 shoot meristem to floral meristem in an angiosperm. In addition, floral organ identity genes encode proteins that determine whether sepals, petals, stamens or carpels are formed during floral development (Yanofsky, supra, 1995; Weigel, Ann. Rev. Genetics 29:19-39 (1995), which is incorporated herein by reference). Some of the floral meristem identity gene products also have a role in specifying floral organ identity.

Floral meristem identity genes have been identified by characterizing genetic mutations that prevent or alter floral meristem formation. Among floral meristem identity gene mutations in Arabidopsis thaliana, those in the gene LEAFY (LFY) generally have the strongest effect on floral meristem identity. Mutations in LFY completely transform the basal-most flowers into secondary shoots and have variable effects on later-arising (apical) flowers. In comparison, mutations in the floral meristem identity gene APETALAl (API) result in replacement of a few basal flowers by inflorescence shoots that are not subtended by leaves.

An apical flower produced in an apl mutant has an indeterminate structure, in which a flower arises within a flower. These mutant phenotypes indicate that both API and LFY contribute to establishing the identity of the floral meristem although neither gene is absolutely required. The phenotype of Ify apl double mutants, in which structures with flower-like characteristics are very rare, indicates that LFY and API encode partially redundant activities.

In addition to the LFY and API genes, a third locus that greatly enhances the apl mutant phenotype has been identified in Arabidopsis. This locus, designated CAULIFLOWER (CAL), derives its name from the resulting WO 97/46079 PCT/US97/09682 9 "cauliflower" phenotype, which is strikingly similar to the common garden variety of cauliflower (Kempin et al., Science 267:522-525 (1995), which is incorporated herein by reference). In an apl cal double mutant, floral meristem behaves as shoot meristem in that there is a massive proliferation of meristems in the position that normally would be occupied by a single flower. However, an Arabidopsis mutant lacking only CAL, such as cal-1, has a normal phenotype, indicating that AP1 can substitute for the loss of CAL in these plants. In addition, because floral meristem that forms in an apl mutant behaves as shoot meristem in an apl cal double mutant, CAL can largely substitute for AP1 in specifying floral meristem. These genetic data indicate that CAL and API encode activities that are partially redundant in converting shoot meristem to floral meristem.

Other genetic loci play at least minor roles in specifying floral meristem identity. For example, although a mutation in APETALA2 (AP2) alone does'not result in altered inflorescence characteristics, ap2 apl double mutants have indeterminate flowers (flowers with shoot-like characteristics; Bowman et al., Development 119:721-743 (1993), which is incorporated herein by reference). Also, mutations in the CLAVATAI (CLV1) gene result in an enlarged meristem and lead to a variety of phenotypes (Clark et al., Development 119:397-418 (1993)). In a clvl apl double mutant, formation of flowers is initiated, but the center of each flower often develops as an indeterminate inflorescence. Thus, mutations in CLAVATA1 result in the loss of floral meristem identity in the center of wild-type flowers.

Genetic evidence also indicates that the gene product of UNUSUAL FLORAL ORGANS (UFO) plays a role in determining the identity of floral meristem. Additional floral WO 97/46079 PCT/US97/09682 meristem identity genes associated with altered floral meristem formation remain to be isolated.

Mutations in another locus, designated TERMINAL FLOWER (TFL), produce phenotypes that generally are reversed as compared to mutations in the floral meristem identity genes. For example, tfl mutants flower early, and the indeterminate apical and lateral meristems develop as determinate floral meristems (Alvarez et al., Plant J. 2:103-116 (1992)). These characteristics indicate that the TFL promotes maintenance of shoot meristem. TFL also acts directly or indirectly to negatively regulate API and LFY expression in shoot meristem since these AP1 and LFY are ectopically expressed in the shoot meristem of tfl mutants (Gustafson-Brown et al., Cell 76:131-143 (1994); Weigel et al., Cell 69:843-859 (1992)). It is recognized that a plant having a mutation in TFL can have a phenotype similar to a non-naturally occurring seed plant of the invention. Such tfl mutants, however, which have a mutation in an endogenous TERMINAL FLOWER gene, are explicitly excluded from the scope of the present invention.

The results of such genetic studies indicate that several floral meristem identity gene products, including AP1, CAL and LFY, act redundantly to convert shoot meristem to floral meristem in an angiosperm. As disclosed herein, ectopic expression of a single floral meristem identity gene product such as AP1, CAL or LFY is sufficient to convert shoot meristem to floral meristem in an angiosperm. Thus, the present invention provides a non-naturally occurring seed plant such as an angiosperm or gymnosperm that contains a first ectopically expressible nucleic acid molecule encoding a first floral meristem identity gene product, provided that such WO 97/46079 PCT/US97/09682 11 ectopic expression is not due to a mutation in an endogenous TERMINAL FLOWER gene.

As disclosed herein, an ectopically expressible nucleic acid molecule encoding a floral meristem identity gene product can be, for example, a transgene encoding a floral meristem identity gene product under control of a heterologous gene regulatory element. In addition, such an ectopically expressible nucleic acid molecule can be an endogenous floral meristem identity gene coding sequence that is placed under control of a heterologous gene regulatory element. The ectopically expressible nucleic acid molecule also can be, for example, an endogenous floral meristem identity gene having a modified gene regulatory element such that the endogenous floral meristem identity gene is no longer subject to negative regulation by TFL.

The term "ectopically expressible" is used herein to refer to a nucleic acid molecule encoding a floral meristem identity gene product that can be expressed in a tissue other than a tissue in which it normally is expressed or at a time other than the time at which it normally is expressed, provided that the floral meristem identity gene product is not expressed from its native, naturally occurring promoter. Ectopic expression of a floral meristem identity gene product is a result of the expression of the gene coding region from a heterologous promoter or from a modified variant of its own promoter, such that expression of the floral meristem identity gene product is no longer in the tissue in which it normally is expressed or at the time at which it normally is expressed. An exogenous nucleic acid molecule encoding an AP1 gene product under control of its native, wild type promoter, for example, does not constitute an ectopically expressible nucleic acid molecule encoding a floral meristem identity gene WO 97/46079 PCT/US97/09682 12 product. However, a nucleic acid molecule encoding an AP1 gene product under control of a constitutive promoter, which results in expression of API in a tissue such as shoot meristem where it is not normally expressed, is an ectopically expressible nucleic acid molecule as defined herein.

Actual ectopic expression of a floral meristem identity gene is dependent on various factors and can be constitutive or inducible expression. For example, AP1, which normally is expressed in floral meristem, is ectopically expressible in the shoot meristem of an angiosperm. As disclosed herein, when a floral meristem identity gene product such as AP1, CAL or LFY is ectopically expressed in shoot meristem in an angiosperm, the shoot meristem is converted to floral meristem and early reproductive development can occur (see Examples I, III and IV).

An ectopically expressible nucleic acid molecule encoding a floral meristem identity gene product can be expressed prior to the time in development at which the corresponding endogenous gene normally is expressed. For example, an Arabidopsis plant grown under continuous light conditions expresses API just prior to day 18, when normal reproductive development (flowering) begins. However, as disclosed herein, API can be ectopically expressed in shoot meristem prior to day 18, resulting in early conversion of shoot meristem to floral meristem and early reproductive development. As disclosed in Example ID, a transgenic Arabidopsis plant that ectopically expresses API in shoot meristem under control of a constitutive promoter can flower at day which is earlier than the time of reproductive development for a non-transgenic plant grown under the same conditions (day 18). It is recognized that in some lb I WO 97/46079 PCT/US97/09682 13 transgenic seed plants containing, for example, an exogenous nucleic acid molecule encoding AP1 under control of a constitutive promoter, neither the exogenous nor endogenous AP1 will be expressed. Such transgenic plants in which API gene expression is cosuppressed, although not characterized by early reproductive development, also can be valuable as disclosed below.

As used herein, the term "floral meristem identity gene product" means a gene product that promotes conversion of shoot meristem to floral meristem in an angiosperm. As disclosed herein in Examples I, II and III, expression of a floral meristem identity gene product such as AP1, CAL or LFY in shoot meristem can convert shoot meristem to floral meristem in an angiosperm. Furthermore, ectopic expression of a floral meristem identity gene product also can promote early reproductive development (see Example ID).

A floral meristem identity gene product is distinguishable from a late flowering gene product or an early flowering gene product. The use of a late flowering gene product or an early flowering gene product is not encompassed within the scope of the present invention. In addition, reference is made herein to an "inactive" floral meristem identity gene product, as exemplified by the product of the Brassica oleracea var.

botrytis CAL gene (BobCAL) (see below). Expression of an inactive floral meristem identity gene product in an angiosperm does not result in the conversion of shoot meristem to floral meristem in the angiosperm. An inactive floral meristem identity gene product such as BobCAL is excluded from the meaning of the term "floral meristem identity gene product" as defined herein.

A floral meristem identity gene product can be, for example, an API gene product having the amino acid WO 97/46079 PCT/US97/09682 14 sequence of SEQ ID NO: 2, which is a 256 amino acid gene product encoded by the Arabidopsis thaliana API cDNA.

The Arabidopsis API cDNA encodes a highly conserved MADS domain, which can function as a DNA-binding domain, and a K domain, which has structural similarity to the coiled-coil domain of keratins and can be involved in protein-protein interactions.

As used herein, the term "APETALA1," "API" or "AP1 gene product" means a floral meristem identity gene product that is characterized, in part, by having an amino acid sequence that has at least about 70 percent amino acid identity with the amino acid sequence of SEQ ID NO: 2 in the region from amino acid 1 to amino acid 163 or with the amino acid sequence of SEQ ID NO: 8 in the region from amino acid 1 to amino acid 163. Like other floral meristem identity gene products, AP1 promotes conversion of shoot meristem to floral meristem in an angiosperm. An AP1 gene product useful in the invention can be, for example, Arabidopsis AP1 having the amino acid sequence of SEQ ID NO: 2; Brassica oleracea API having the amino acid sequence of SEQ ID NO: 4; Brassica oleracea var. botrytis AP1 having the amino acid sequence of SEQ ID NO: 6 or Zea mays API having the amino acid sequence of SEQ ID NO: 8.

In wild-type Arabidopsis, API RNA is expressed in flowers but is not detectable in roots, stems or leaves (Mandel et al., Nature 360:273-277 (1992), which is incorporated herein by reference). The earliest detectable expression of API RNA is in young floral meristem at the time it initially forms on the flanks of shoot meristem. Expression of API increases as the floral meristem increases in size; no API expression is detectable in shoot meristem. In later stages of WO 97/46079 PCT/US97/09682 development, API expression ceases in cells that will give rise to reproductive organs of a flower (stamens and carpels), but is maintained in cells that will give rise to non-reproductive organs (sepals and petals; Mandel, supra, 1992). Thus, in nature, API expression is restricted to floral meristem and to certain regions of the flowers that develop from this meristemic tissue.

CAULIFLOWER (CAL) is another example of a floral meristem identity gene product. As used herein, the term "CAULIFLOWER," "CAL" or "CAL gene product" means a floral meristem identity gene product that is characterized, in part, by having an amino acid sequence that has at least about 70 percent amino acid identity with the amino acid sequence of SEQ ID NO: 10 in the region from amino acid 1 to amino acid 160 or with the amino acid sequence of SEQ ID NO: 12 in the region from amino acid 1 to amino acid 160.

A CAL gene product is exemplified by the Arabidopsis CAL gene product, which has the amino acid sequence of SEQ ID NO: 10, or the Brassica oleracea CAL gene product, which has the amino acid sequence of SEQ ID NO: 12. As disclosed herein, CAL, like AP1, contains a MADS domain and a K domain. The MADS domains of CAL and AP1 differ in only five of 56 amino acid residues, where four of the five differences represent conservative amino acid replacements. Over the entire sequence, the Arabidopsis CAL and Arabidopsis AP1 sequences (SEQ ID NOS: 10 and 2) are 76% identical and are 88% similar if conservative amino acid substitutions are allowed.

Similar to the expression pattern of API, CAL RNA is expressed in young floral meristem in Arabidopsis.

However, in contrast to API expression, which is high throughout sepal and petal development, CAL expression is WO 97/46079 PCT/US97/09682 16 low in these organs. Thus, in nature, CAL is expressed in floral meristem and, to a lesser extent, in the organs of developed flowers.

The skilled artisan will recognize that, due to the high sequence conservation between AP1 and CAL, a novel ortholog can be categorized as both a CAL and an AP1, as defined herein. However, if desired, an AP1 ortholog can be distinguished from a CAL ortholog by demonstrating a greater similarity to Arabidopsis API than to any other MADS box gene, such as CAL, as set forth in Purugganan et al. (Genetics 140:345-356 (1995), which is incorporated herein by reference). Furthermore, AP1 can be distinguished from CAL by its ability to complement, or restore a wild-type phenotype, when introduced into a strong apl mutant. For example, introduction of Arabidopsis API (AGL7) complements the phenotype of the strong apl-I mutant; however, introduction of CAL (AGL10) into a cal-i apl-i mutant plant yields the apl-1 single mutant phenotype, demonstrating that CAL cannot complement the apl-1 mutation (see, for example, Mandel et al., supra, 1992; Kempin et al., supra, 1995). Thus, AP1 can be distinguished from CAL, if desired, by the ability of a nucleic acid molecule encoding AP1 to complement a strong apl mutant such as apl-i or LEAFY (LFY) is yet another example of a floral meristem identity gene product. As used herein, the term "LEAFY" or "LFY" or "LFY gene product" means a floral meristem identity gene product that is characterized, in part, by having an amino acid sequence that has at least about 70 percent amino acid identity with the amino acid sequence of SEQ ID NO: 16. In nature, LFY is expressed in floral meristem as well as during vegetative WO 97/46079 PCT/US97/09682 17 development. As disclosed herein, ectopic expression in shoot meristem of a floral meristem identity gene product, which normally is expressed in floral meristem, can convert shoot meristem to floral meristem in an angiosperm. Under appropriate conditions, ectopic expression in shoot meristem of a floral meristem identity gene product such as AP1, CAL, LFY, or a combination thereof, can promote early reproductive development.

Flower development in Arabidopsis is recognized in the art as a model for flower development in angiosperms in general. Gene orthologs corresponding to the Arabidopsis genes involved in the early steps of flower formation have been identified in distantly related angiosperm species, and these gene orthologs show remarkably similar patterns of RNA expression. Mutations in gene orthologs also result in phenotypes that correspond to the phenotype produced by a similar mutation in Arabidopsis. For example, orthologs of the Arabidopsis floral meristem identity genes API and LFY and the Arabidopsis organ identity genes AGAMOUS, APETALA3 and PISTILLATA have been isolated from monocots such as maize and, where characterized, reveal the anticipated RNA expression patterns and related mutant phenotypes (Schmidt et al., Plant Cell 5:729-737 (1993); and Veit et al., Plant Cell 5:1205-1215 (1993), each of which is incorporated herein by reference). Furthermore, a gene ortholog can be functionally interchangeable in that it can function across distantly related species boundaries (Mandel et al., Cell 71:133-143 (1992), which is incorporated herein by reference). Taken together, these data suggest that the underlying mechanisms controlling the initiation and proper development of flowers are conserved across distantly related dicot and monocot boundaries.

WO 97/46079 PCT/US97/09682 18 Floral meristem identity genes in particular are conserved among distantly related angiosperm and gymnosperm species. For example, a gene ortholog of Arabidopsis API has been isolated from Antirrhinum majus (snapdragon; Huijser et al., EMBO J. 11:1239-1249 (1992), which is incorporated herein by reference). As disclosed herein, an ortholog of Arabidopsis API also has been isolated from Brassica oleracea var. botrytis (cauliflower) and Zea Mays (maize; see Example VA).

Furthermore, API orthlogs also can be isolated from gymnosperms. Similarly, gene orthologs of Arabidopsis LFY have been isolated from angiosperms such as Antirrhinum majus, tobacco and poplar tree and from gymnosperms such as Douglas fir (Coen et al., Cell, 63:1311-1322 (1990); Kelly et al., Plant Cell 7:225-234 (1995); and Rottmann et al., Cell Biochem. Suppl. 17B: 23 (1993); Strauss et al., Molec. Breed 1:5-26 (1995), each of which is incorporated herein by reference). The conservation of floral meristem identity gene products in non-flowering plants such as coniferous trees indicates that floral meristem identity genes can promote the reproductive development of gymnosperms as well as angiosperms.

The characterization of apl and Ify mutants also indicates that floral meristem identity gene products such as API and LFY function similarly in distantly related plant species. For example, a mutation in the Antirrhinum API ortholog results in a phenotype similar to the Arabidopsis apl indeterminate flower within a flower phenotype (Huijser et al., supra, 1992).

In addition, a mutation in the Antirrhinum LFY ortholog results in a phenotype similar to the Arabidopsis Ify mutant phenotype (Coen et al., supra, 1995) WO 97/46079 PCT/US97/09682 19 A floral meristem identity gene product also can function across species boundaries. For example, introduction of a nucleic acid molecule encoding Arabidopsis LFY into a heterologous seed plant such as tobacco or aspen results in early reproductive development (Weigel and Nilsson, Nature 377:495-500 (1995), which is incorporated herein by reference). As disclosed herein, a nucleic acid molecule encoding an Arabidopsis AP1 gene product (SEQ ID NO: 1) or an Arabidopsis CAL gene product (SEQ ID NO: 9) can be introduced into a heterologous seed plant such as corn, wheat, rice or pine and, upon ectopic expression, can promote early reproductive development in the transgenic seed plant. Furthermore, as disclosed herein, the conserved nature of the API, CAL and LFY coding sequences among diverse seed plant species allows a nucleic acid molecule encoding a floral meristem identity gene product isolated from essentially any seed plant to be introduced into essentially any other seed plant, wherein, upon appropriate expression of the introduced nucleic acid molecule in the seed plant, the floral meristem identity gene product promotes early reproductive development in the seed plant.

If desired, a novel API, CAL or LFY coding sequence can be isolated from a seed plant using a nucleotide sequence as a probe and methods well known in the art of molecular biology (Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual (Second Edition), Plainview, NY: Cold Spring Harbor Laboratory Press (1989), which is incorporated herein by reference). As exemplified herein and discussed in detail below (see Example VA), an API ortholog from Zea Mays (maize; SEQ ID NO: 7) was isolated using the Arabidopsis API cDNA (SEQ ID NO: 1) as a probe.

In general terms, the invention also relates to a non-naturally occurring seed plant that contains a first ectopically expressible nucleic acid molecule encoding a floral meristem identity gene product, provided that the first nucleic acid molecule is not ectopically expressed due to a mutation in an endogenous TERMINAL FLOWER gene, and that is characterized by early reproductive development. As used herein, the term "characterized by early reproductive development," when used in reference to a non-naturally occurring seed plant of the invention, means a non-naturally occurring seed plant that forms reproductive structures earlier than the time when reproductive structures form on a corresponding naturally occurring seed plant that is grown under the same conditions and that does not ectopically express a floral meristem identity gene product. For example, the reproductive structure of an angiosperm is a flower, and the reproductive structure of a coniferous plant is a cone. For a particular naturally occurring seed plant, reproductive development occurs at a well-defined time that depends, in part, on genetic factors as well as on, environmental conditions, such as day length and temperature. Thus, given a defined set of environmental condition and lacking ectopic expression of a floral meristem identity gene product, a naturally occurring seed plant will undergo reproductive development at a relatively fixed time.

It is recognized that various transgenic plants that are characterized by early reproductive development have been described previously. Such transgenic plants, as discussed herein, are distinguishable from a non-naturally occurring seed plant of the invention or are explicitly excluded from the present invention. The product of a "late-flowering gene" can promote early reproductive development. However, a late flowering gene product is not a floral meristem identity gene product ",BP 1: I dh.,m I 1 I1t 07/00

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WO 97/46079 PCT/US97/09682 21 since it does not specify the conversion of shoot meristem to floral meristem in an angiosperm. Therefore, a transgenic plant expressing a late-flowering gene product is distinguishable from a non-naturally occurring seed plant of the invention. For example, a transgenic plant expressing the late-flowering gene, CONSTANS (CO), flowers earlier than the corresponding wild type plant, but does not contain an ectopically expressible nucleic acid molecule encoding a floral meristem identity gene product (Putterill et al., Cell 80:847-857 (1995)).

Thus, the early-flowering transgenic plant described by Putterill et al. is not a non-naturally occurring seed plant as defined herein.

Early reproductive development also has been observed in a transgenic tobacco plant expressing an exogenous rice MADS domain gene. Although the product of the rice MADS domain gene promotes early reproductive development, it does not specify the identity of floral meristem and, thus, cannot convert shoot meristem to floral meristem in an angiosperm (Chung et al., Plant Mol. Biol. 26:657-665 (1994)). Therefore, an early-flowering transgenic plant containing this rice MADS domain gene, like an early-flowering transgenic plant containing CONSTANS, is distinguishable from an early-flowering non-naturally occurring seed plant of the invention.

Mutations in a class of genes known as "early-flowering genes" also produce plants characterized by early reproductive development. Such early-flowering genes include, for example, EARLY FLOWERING 1-3 (ELF1, ELF2, ELF3); EMBRYONIC FLOWER 1,2 (EMF1, EMF2); LONG HYPOCOTYL 1,2 (HY1, HY2); PHYTOCHROME B (PHYB), SPINDLY (SPY) and TERMINAL FLOWER (TFL) (Weigel, supra, 1995).

The wild type product of an early-flowering gene retards WO 97/46079 PCTIUS97/09682 22 reproductive development and is distinguishable from a floral meristem identity gene product in that an early-flowering gene product does not promote conversion of shoot meristem to floral meristem in an angiosperm. A plant that flowers early due to the loss of an early-flowering gene product function is distinct from a non-naturally occurring seed plant of the invention characterized by early reproductive development since such a plant does not contain an ectopically expressible nucleic acid molecule encoding a floral meristem identity gene product.

An Arabidopsis plant having a mutation in the TERMINAL FLOWER (TFL) gene is characterized by early reproductive development and by the conversion of shoots to flowers (Alvarez et al., Plant J. 2:103-116 (1992), which is incorporated herein by reference). However, TFL is not a floral meristem identity gene product, as defined herein. Specifically, it is the loss of TFL that promotes conversion of shoot meristem to floral meristem.

Since the function of TFL is to antagonize formation of floral meristem, a tfl mutant, which lacks functional TFL, converts shoot meristem to floral meristem prematurely. Although TFL is not a floral meristem identity gene product and does not itself convert shoot meristem to floral meristem, the loss of TFL can result in a plant with an ectopically expressed floral meristem identity gene product. However, such a tfl mutant, in which a mutation in an endogenous TERMINAL FLOWER gene results in conversion of shoot meristem to floral meristem, is excluded explicitly from the present invention.

As used herein, the term "transgenic" refers to a seed plant that contains in its genome an exogenous nucleic acid molecule, which can be derived from the same WO 97/46079 PCTIUS97/09682 23 or a different plant species. The exogenous nucleic acid molecule can be a gene regulatory element such as a promoter, enhancer or other regulatory element or can contain a coding sequence, which can be linked to a heterologous gene regulatory element.

As used herein, the term "seed plant" means an angiosperm or a gymnosperm. The term "angiosperm," as used herein, means a seed-bearing plant whose seeds are borne in a mature ovary (fruit). An angiosperm commonly is recognized as a flowering plant. The term "gymnosperm," as used herein, means a seed-bearing plant with seeds not enclosed in an ovary.

Angiosperms are divided into two broad classes based on the number of cotyledons, which are seed leaves that generally store or absorb food. Thus, a monocotyledonous angiosperm is an angiosperm having a single cotyledon, and a dicotyledonous angiosperm is an angiosperm having two cotyledons. Angiosperms are well known and produce a variety of useful products including materials such as lumber, rubber, and paper; fibers such as cotton and linen; herbs and medicines such as quinine and vinblastine; ornamental flowers such as roses and orchids; and foodstuffs such as grains, oils, fruits and vegetables.

Angiosperms encompass a variety of flowering plants, including, for example, cereal plants, leguminous plants, oilseed plants, hardwood trees, fruit-bearing plants and ornamental flowers, which general classes are not necessarily exclusive. Such angiosperms include for example, a cereal plant, which produces an edible grain cereal. Such cereal plants include, for example, corn, rice, wheat, barley, oat, rye, orchardgrass, guinea grass, sorghum and turfgrass. In addition, a leguminous plant is an angiosperm that is a member of the pea family WO 97/46079 PCT/US97/09682 24 (Fabaceae) and produces a characteristic fruit known as a legume. Examples of leguminous plants include, for example, soybean, pea, chickpea, moth bean, broad bean, kidney bean, lima bean, lentil, cowpea, dry bean, and peanut. Examples of legumes also include alfalfa, birdsfoot trefoil, clover and sainfoin. An oilseed plant also is an angiosperm with seeds that are useful as a source of oil. Examples of oilseed plants include soybean, sunflower, rapeseed and cottonseed.

An angiosperm also can be a hardwood tree, which is a perennial woody plant that generally has a single stem (trunk). Examples of such trees include alder, ash, aspen, basswood (linden), beech, birch, cherry, cottonwood, elm, eucalyptus, hickory, locust, maple, oak, persimmon, poplar, sycamore, walnut and willow. Trees are useful, for example, as a source of pulp, paper, structural material and fuel.

An angiosperm also can be a fruit-bearing plant, which produces a mature, ripened ovary (usually containing seeds) that is suitable for human or animal consumption. For example, hops are a member of the mulberry family prized for their flavoring in malt liquor. Fruit-bearing angiosperms also include grape, orange, lemon, grapefruit, avocado, date, peach, cherry, olive, plum, coconut, apple and pear trees and blackberry, blueberry, raspberry, strawberry, pineapple, tomato, cucumber and eggplant plants. An ornamental flower is an angiosperm cultivated for its decorative flower. Examples of commercially important ornamental flowers include rose, orchid, lily, tulip and chrysanthemum, snapdragon, camellia, carnation and petunia plants. The skilled artisan will recognize that the methods of the invention can be practiced using these or other angiosperms, as desired.

25 Gymnosperms encompass four divisions: cycads, ginkgo, conifers and gnetophytes. The conifers are the most widespread of living gymnosperms and frequently are cultivated for structural wood or for pulp or paper.

Conifers include redwood trees, pines, firs, spruces, hemlocks, Douglas firs, cypresses, junipers and yews.

The skilled artisan will recognize that the methods of the invention can be practiced with these and other gymnosperms.

As used herein, the term "non-naturally occurring seed plant" means a seed plant containing a genome that has been modified by man. A transgenic seed plant, for example, is a non-naturally occurring seed plant that contains an exogenous nucleic acid molecule and, therefore, has a genome that has been modified by man. Furthermore, a seed plant that contains, for example, a mutation in an endogenous floral meristem identity gene regulatory element as a result of calculated exposure to a mutagenic agent also contains a S: genome that has been modified by man. In contrast, a seed plant containing a spontaneous or naturally :::occurring mutation is not a "non-naturally occurring seed plant" and, therefore, is not encompassed within the 25 invention.

In general terms, the invention relates to a non-naturally occurring seed plant containing a first ectopically expressible nucleic acid molecule 30 encoding a first floral meristem identity gene product, provided that the first nucleic acid molecule is not ectopically expressed due to a mutation in an endogenous o TERMINAL FLOWER gene. If desired, a non-naturally occurring seed plant of the invention can contain a second ectopically expressible nucleic acid molecule encoding a second floral meristem identity gene product S 1 that is different from the first floral meristem identity IS \h Is III, III\ S11- I\ 32967 19 *7i00 WO 97/46079 PCT/US97/09682 26 gene product, provided that the first or second nucleic acid molecule is not ectopically expressed due to a mutation in an endogenous TERMINAL FLOWER gene.

An ectopically expressible nucleic acid molecule encoding a floral meristem identity gene product can be expressed, as desired, either constitutively or inducibly. Such an ectopically expressible nucleic acid molecule encoding a floral meristem identity gene product can be an endogenous floral meristem identity gene that has, for example, a mutation in a gene regulatory element. An ectopically expressible nucleic acid molecule encoding a floral meristem identity gene product also can be an endogenous nucleic acid molecule encoding a floral meristem identity gene product that is linked to an exogenous, heterologous gene regulatory element that confers ectopic expression. In addition, an ectopically expressible nucleic acid molecule encoding a floral meristem identity gene product can be an exogenous nucleic acid molecule that encodes a floral meristem identity gene product under control of a heterologous gene regulatory element.

A non-naturally occurring seed plant of the invention can contain an endogenous floral meristem identity gene having a modified gene regulatory element.

The term "modified gene regulatory element," as used herein in reference to the regulatory element of a floral meristem identity gene, means a regulatory element having a mutation that results in ectopic expression of the linked endogenous floral meristem identity gene. Such a gene regulatory element can be, for example, a promoter or enhancer element and can be positioned 5' or 3' to the coding sequence or within an intronic sequence of the floral meristem identity gene. A modified gene regulatory element can have, for example, a nucleotide insertion, deletion or substitution that is produced, for 27 example, by chemical mutagenesis using a mutagen such as ethylmethane sulfonate or by insertional mutagenesis using a transposable element. A modified gene regulatory element can be a functionally inactivated binding site for TFL or a functionally inactivated binding site for a gene product regulated by TFL, such that modification of the gene regulatory element results in ectopic expression of the linked floral meristem identity gene product, for example, in the shoot meristem of an angiosperm.

In general terms, the present invention also relates to a transgenic seed plant containing a first exogenous gene promoter that regulates a first ectopically expressible nucleic acid molecule encoding a first floral meristem identity gene product and a second exogenous gene promoter that regulates a second ectopically expressible nucleic acid molecule encoding a second floral meristem identity gene product.

In general terms, the present invention also relates to a transgenic seed plant containing a first exogenous S.ectopically expressible nucleic acid molecule encoding a first floral meristem identity gene product and a second exogenous gene promoter that regulates a second ectopically expressible nucleic acid molecule encoding a second floral meristem identity gene product, provided that the first nucleic acid molecule is not ectopically expressed due to a mutation in an endogenous

TERMINAL

FLOWER gene.

30 In general terms, the invention relates to a transgenic seed plant containing a first exogenous ectopically expressible nucleic acid molecule encoding a first floral meristem identity gene product, provided that the first second nucleic acid molecule is not ectopically expressed due to a mutation in an endogenous TERMINAL FLOWER gene, and further containing a second '0 PP. IS I I -1-1H\Sllel t 02967 C10 91,07/01 WO 97/46079 PCT/US97/09682 28 exogenous ectopically expressible nucleic acid molecule encoding a second floral meristem identity gene product, where the first floral meristem identity gene product is different from the second floral meristem identity gene product.

As disclosed herein, ectopic expression of two different floral meristem identity gene products can be particularly useful. A transgenic Arabidopsis line constitutively expressing API under control of the cauliflower mosaic virus 35S promoter (see Example I) was crossed with a transgenic Arabidopsis line constitutively expressing LFY under control of the cauliflower mosaic virus 35S promoter (see Example III), and the resulting progeny were analyzed. A fraction of the progeny flowered were characterized by enhanced early reproductive development as compared to the early reproductive development of 35S-API transgenic lines or transgenic lines. PCR-based analyses demonstrated that all of the transgenic plants that were characterized by enhanced early reproductive development contained both the 35S-API and 35S-LFY transgenes. These results indicate that ectopic expression of the combination of AP1 and LFY in a seed plant can result in enhanced early reproductive development as compared to the early reproductive development obtained by ectopic expression of AP1 or LFY alone. Thus, by using a combination of two different floral meristem identity gene products, plant breeding, for example, can be accelerated further as compared to the use of a single floral meristem identity gene product.

A useful combination of first and second floral meristem identity gene products can be, for example, AP1 and LFY, CAL and LFY, or AP1 and CAL. A particularly useful combination of first and second floral meristem 29 identity gene products is the combination of AP1 with LFY, as disclosed above, or the combination of CAL with LFY. Where a transgenic seed plant of the invention contains first and second exogenous nucleic acid molecules encoding different floral meristem identity gene products, it will be recognized that the order of introducing the first and second nucleic acid molecules into the seed plant is not important for purposes of the present invention. Thus, a transgenic seed plant of the invention having, for example, API as a first floral meristem identity gene product and LFY as a second floral meristem identity gene product is equivalent to a transgenic seed plant having LFY as a first floral meristem identity gene product and API as a second floral meristem identity gene product.

Also provided are methods of converting shoot meristem to floral meristem in an angiosperm by ectopically expressing a first ectopically expressible nucleic acid molecule encoding a first floral meristem identity gene product in the angiosperm. Thus, the invention provides, for example, a method of converting shoot meristem to floral meristem in an angiosperm by introducing an exogenous, ectopically expressible nucleic acid molecule encoding a floral meristem identity gene product into the angiosperm, thereby producing a transgenic angiosperm. A floral meristem identity gene product such as AP1, CAL or LFY, or a chimeric protein containing, in part, a floral meristem identity gene product, as disclosed below, is useful in converting shoot meristem to floral meristem.

9s As used herein, the term "introducing," when used in reference to a nucleic acid molecule and a seed plant such as an angiosperm or a gymnosperm, means transferring an exogenous nucleic acid molecule into the P RIS Ih I Is.,I,e lH\Spci\ 3'6 .dOC L9,07,O00 WO 97/46079 PCT/US97/09682 seed plant. For example, an exogenous nucleic acid molecule encoding a floral meristem identity gene product can be introduced into a seed plant by a variety of methods including Agrobacterium-mediated transformation or direct gene transfer methods such as electroporation or microprojectile-mediated transformation.

Transformation methods based upon the soil bacterium Agrobacterium tumefaciens, known as "agro-infection," are useful for introducing a nucleic acid molecule into a broad range of angiosperms and gymnosperms. The wild type form of Agrobacterium contains a Ti (tumor-inducing) plasmid that directs production of tumorigenic crown gall growth on host plants. Transfer of the tumor-inducing T-DNA region of the Ti plasmid to a plant genome requires the Ti plasmid-encoded virulence genes as well as T-DNA borders, which are a set of direct DNA repeats that delineate the region to be transferred. Agrobacterium-based vector is a modified form of a Ti plasmid, in which the tumor inducing functions are replaced by nucleic acid sequence of interest to be introduced into the plant host.

Current protocols for Agrobacterium-mediated transformation employ cointegrate vectors or, preferably, binary vector systems in which the components of the Ti plasmid are divided between a helper vector, which resides permanently in the Agrobacterium host and carries the virulence genes, and a shuttle vector, which contains the gene of interest bounded by T-DNA sequences. A variety of binary vectors are well known in the art and are commercially available from, for example, Clontech (Palo Alto, California). Methods of coculturing Agrobacterium with cultured plant cells or wounded tissue such as leaf tissue, root explants, hypocotyledons, stem pieces or tubers, for example, also are well known in the WO 97/46079 PCTIUS97/09682 31 art (Glick and Thompson Methods in Plant Molecular Biology and Biotechnology, Boca Raton, FL: CRC Press (1993), which is incorporated herein by reference).

Wounded cells within the plant tissue that have been infected by Agrobacterium can develop organs de novo when cultured under the appropriate conditions; the resulting transgenic shoots eventually give rise to transgenic plants containing the exogenous nucleic acid molecule of interest, as described in Example I.

Agrobacterium-mediated transformation has been used to produce a variety of transgenic seed plants (see, for example, Wang et al. (eds), Transformation of Plants and Soil Microorganisms, Cambridge, UK: University Press (1995), which is incorporated herein by reference). For example, Agrobacterium-mediated transformation can be used to produce transgenic crudiferous plants such as Arabidopsis, mustard, rapeseed and flax; transgenic leguminous plants such as alfalfa, pea, soybean, trefoil and white clover; and transgenic solanaceous plants such as eggplant, petunia, potato, tobacco and tomato. In addition, Agrobacterium-mediated transformation can be used to introduce exogenous nucleic acids into apple, aspen, belladonna, black currant, carrot, celery, cotton, cucumber, grape, horseradish, lettuce, morning glory, muskmelon, neem, poplar, strawberry, sugar beet, sunflower, walnut and asparagus plants (see, for example, Glick and Thompson, supra, 1993).

Microprojectile-mediated transformation also is a well known method of introducing an exogenous nucleic acid molecule into a variety of seed plant species. This method, first described by Klein et al., Nature 327:70-73 (1987), which is incorporated herein by reference, relies on microprojectiles such as gold or tungsten that are coated with the desired nucleic acid molecule by WO 97/46079 PCT/US97/09682 32 precipitation with calcium chloride, spermidine or PEG.

The microprojectile particles are accelerated at high speed into seed plant tissue using a device such as the Biolistic TM PD-1000 (Biorad, Hercules, California).

Microprojectile-mediated delivery or "particle bombardment" is especially useful to transform seed plants that are difficult to transform or regenerate using other methods. Microprojectile-mediated transformation has been used, for example, to generate a variety of transgenic seed plant species, including cotton, tobacco, corn, hybrid poplar and papaya (see, for example, Glick and Thompson, supra, 1993). The transformation of important cereal crops such as wheat, oat, barley, sorghum and rice also has been achieved using microprojectile-mediated delivery (Duan et al., Nature Biotech. 14:494-498 (1996); Shimamoto, Curr. Opin.

Biotech. 5:158-162 (1994), each of which is incorporated herein by reference). A rapid transformation regeneration system for the production of transgenic plants, such as transgenic wheat, in two to three months also can be useful in producing a transgenic seed plant of the invention (European Patent No. EP 0 709 462 A2, Application number 95870117.9, filed 25 October 1995, which is incorporated herein by reference).

Thus, a variety of methods for introducing a nucleic acid molecule into a seed plant are well known in the art. Important crop species such as rice, for example, have been transformed using microprojectile delivery, Agrobacterium-mediated transformation or protoplast transformation (Hiei et al., The Plant J.

6(2):271-282 (1994); Shimamoto, Science 270:1772-1773 (1995), each of which is incorporated herein by reference). Fertile transgenic maize has been obtained, for example, by microparticle bombardment (see Wang et al., supra, 1995). As discussed above, barley, wheat, WO 97/46079 PCT/US97/09682 33 oat and other small-grain cereal crops also have been transformed, for example, using microparticle bombardment (see Wang et al., supra, 1995).

Methods of transforming forest trees including both angiosperms and gymnosperms also are well known in the art. Transgenic angiosperms such as members of the genus Populus, which includes aspens and poplars, have been generated using Agrobacterium-mediated transformation, for example. In addition, transgenic Populus and sweetgum, which are of interest for biomass production for fuel, also have been produced. Transgenic gymnosperms, including conifers such as white spruce and larch, also have been obtained, for example, using microprojectile bombardment (Wang et al., supra, 1995).

The skilled artisan will recognize that Agrobacterium-mediated or microprojectile-mediated transformation, as disclosed herein, or other methods known in the art can be used to introduce a nucleic acid molecule encoding a floral meristem identity gene product into a seed plant according to the methods of the invention.

The term "converting shoot meristem to floral meristem," as used herein, means promoting the formation of flower progenitor tissue where shoot progenitor tissue otherwise would be formed in the angiosperm. As a result of the conversion of shoot meristem to floral meristem, flowers form in an angiosperm where shoots normally would form. The conversion of shoot meristem to floral meristem can be identified using well known methods, such as scanning electron microscopy, light microscopy or visual inspection (see, for example, Mandel and Yanofsky, Plant Cell 7:1763-1771 (1995), which is incorporated herein by reference or Weigel and Nilsson, supra, 1995).

WO 97/46079 PCT/US97/09682 34.

Provided herein are methods of converting shoot meristem to floral meristem in an angiosperm by introducing a first ectopically expressible nucleic acid molecule encoding a first floral meristem identity gene product and a second ectopically expressible nucleic acid molecule encoding a second floral meristem identity gene product into the angiosperm, where the first floral meristem identity gene product is different from the second floral meristem identity gene product. As discussed above, first and second floral meristem identity gene products useful in converting shoot meristem to floral meristem in an angiosperm can be, for example, AP1 and LFY, CAL and LFY, or API and CAL.

Also provided herein are methods of promoting early reproductive development in a seed plant by ectopically expressing a first nucleic acid molecule encoding a first floral meristem identity gene product in the seed plant, provided that the first nucleic acid molecule is not ectopically expressed due to a mutation in an endogenous TERMINAL FLOWER gene. For example, the invention provides a method of promoting early reproductive development in a seed plant by introducing an ectopically expressible nucleic acid molecule encoding a floral meristem identity gene product into the seed plant, thus producing a transgenic seed plant. A floral meristem identity gene product such as AP1, CAL or LFY, or a chimeric protein containing, in part, a floral meristem identity gene product, as disclosed below, is useful in methods of promoting early reproductive development.

The term "promoting early reproductive development," as used herein in reference to a seed plant, means promoting the formation of a reproductive structure earlier than the time when a reproductive structure would form on a corresponding seed plant that WO 97/46079 PCT/US97/09682 is grown under the same conditions and that does not ectopically express a floral meristem identity gene product. As discussed above, the time when reproductive structures form on a particular seed plant that does not ectopically express a floral meristem identity gene product is relatively fixed and depends, in part, on genetic factors as well as environmental conditions, such as day length and temperature. Thus, given a defined set of environmental conditions, a naturally occurring angiosperm, for example, will flower at a relatively fixed time. Similarly, given a defined set of environmental conditions, a naturally occurring coniferous gymnosperm, for example, will produce cones at a relatively fixed time.

As disclosed herein, ectopic expression of a nucleic acid molecule encoding a floral meristem identity gene product in an angiosperm converts shoot meristem to floral meristem in the angiosperm. Furthermore, ectopic expression of a nucleic acid molecule encoding a floral meristem identity gene product such as AP1, CAL or LFY in an angiosperm prior to the time when endogenous floral meristem identity gene products are expressed in the angiosperm can convert shoot meristem to floral meristem precociously, resulting in early reproductive development in the angiosperm, as indicated by early flowering. In the same manner, ectopic expression of a nucleic acid molecule encoding AP1, CAL, or LFY, for example, in a gymnosperm prior to the time when endogenous floral meristem identity gene products are expressed in the gymnosperm results in early reproductive development in the gymnosperm.

For a given seed plant species and particular set of growth conditions, constitutive expression of a floral meristem identity gene product results in a relatively invariant time of early reproductive WO 97/46079 PCT/US97/09682 36 development, which is the earliest time when all factors necessary for reproductive development are active. For example, as shown in Example ID, constitutive expression of API in transgenic Arabidopsis plants grown under "long-day" light conditions results in early reproductive development, at day 10 as compared to the normal time of reproductive development, which is day 18 in non-transgenic Arabidopsis plants grown under the same conditions. Thus, under these conditions, day 10 is the relatively invariant time of early reproductive development for Arabidopsis transgenics that constitutively express a floral meristem identity gene product.

However, in addition to methods of constitutively expressing a floral meristem identity gene product, the present invention provides methods of selecting the time of early reproductive development. As disclosed herein, floral meristem gene product expression or activity can be regulated in response to an inducing agent or cognate ligand, for example, such that the time of early reproductive development can be selected. For example, in Arabidopsis transgenics grown under the conditions described above, the time of early reproductive development need not necessarily be the relatively invariant day 10 at which early reproductive development occurs as a consequence of constitutive floral meristem identity gene product expression. If floral meristem identity gene product expression is rendered dependent upon the presence of an inducing agent, early reproductive development can be selected to occur, for example, on day 14, by contacting the seed plant with an inducing agent on or slightly before day 14.

Thus, the present invention provides recombinant nucleic acid molecules, transgenic seed plant containing such recombinant nucleic acid molecules and methods for selecting the time of early reproductive development. These methods allow a farmer or horticulturist, for example, to determine the time of early reproductive development. The methods of the invention can be useful, for example, in allowing a grower to respond to an approaching storm or impending snap-freeze by selecting the time of early reproductive development such that the crop can be harvested before being harmed by the adverse weather conditions. The methods of the invention for selecting the time of early reproductive development also can be useful to spread out the time period over which transgenic seed plants are ready to be harvested. For example, the methods of the invention can be used to increase floral meristem identity gene product expression in different crop fields at different times, resulting in a staggered time of harvest for the different fields.

Thus, the present invention provides a o: recombinant nucleic acid molecule containing an inducible 25 regulatory element operably linked to a nucleic acid molecule encoding CAULIFLOWER (CAL). As disclosed herein, a recombinant nucleic acid molecule of the invention can contain an inducible regulatory element such as a copper inducible element, tetracycline inducible element, 30 ecdysone inducible element or heat shock inducible element.

e* o*o *06 F I- I I I I% 1 7 c 19 OO 0 38 The invention also provides a transgenic seed plant containing a recombinant nucleic acid molecule comprising an inducible regulatory element operably linked to a nucleic acid molecule encoding CAL. Such a transgenic seed plant can be an angiosperm or gymnosperm.

A transgenic seed plant of the invention can contain a recombinant nucleic acid molecule comprising a copper inducible element tetracycline inducible element, ecdysone inducible element or heat-shock inducible element operably linked to a nucleic acid molecule encoding CAL.

The term "recombinant nucleic acid molecule," as used herein, means a non-naturally occurring nucleic acid molecule that has been manipulated in vitro such that it is genetically distinguishable from a naturally occurring nucleic acid molecule. A recombinant nucleic acid molecule of the inventioncomprises two nucleic acid molecules that have been manipulated in vitro such that the two nucleic acid molecules are operably linked.

4 4 e 4 0 *o4 *t 4 4 4 *e ,;BPISl\l~om~i\ [s.lhelHI'V~'-i i rl.:.e L9 n3 WO 97/46079 PCT/US97/09682 39 As used herein, the term "inducible regulatory element" means a nucleic acid molecule that confers conditional expression upon an operably linked nucleic acid molecule, where expression of the operably linked nucleic acid molecule is increased in the presence of a particular inducing agent as compared to expression of the nucleic acid molecule in the absence of the inducing agent. In a method of the invention, a useful inducible regulatory element has the following characteristics: confers low level expression upon an operably linked nucleic acid molecule in the absence of an inducing agent; confers high level expression upon an operably linked nucleic acid molecule in the presence of an appropriate inducing agent; and utilizes an inducing agent that does not interfere substantially with the normal physiology of a transgenic seed plant treated with the inducing agent. It is recognized, for example, that, subsequent to introduction into a seed plant, a particularly useful inducible regulatory element is one that confers an extremely low level of expression upon an operably linked nucleic acid molecule in the absence of inducing agent. Such an inducible regulatory element is considered to be tightly regulated.

The term "operably linked," as used in reference to a regulatory element, such as a promoter or inducible regulatory element, and a nucleic acid molecule encoding a floral meristem identity gene product, means that the regulatory element confers regulated expression upon the operably linked nucleic acid molecule encoding the floral meristem identity gene product. Thus, the term operably linked, as used herein in reference to an inducible regulatory element and a nucleic acid molecule encoding a floral meristem identity gene product, means that the inducible regulatory element is linked to the nucleic acid molecule encoding a floral meristem identity gene product such that the inducible regulatory element WO 97/46079 PCT/US97/09682 increases expression of the floral meristem identity gene product in the presence of the appropriate inducing agent. It is recognized that two nucleic acid molecules that are operably linked contain, at a minimum, all elements essential for transcription, including, for example, a TATA box. One skilled in the art knows, for example, that an inducible regulatory element that lacks minimal promoter elements can be combined with a nucleic acid molecule having minimal promoter elements and a nucleic acid molecule encoding a floral meristem identity gene product such that expression of the floral meristem identity gene product can be increased in the presence of the appropriate inducing agent.

A particularly useful inducible regulatory element can be, for example, a copper-inducible promoter (Mett et al., Proc. Natl. Acad. Sci. USA 90:4567-4571 (1993), which is incorporated herein by reference); tetracycline-inducible regulatory element (Gatz et al., Plant J. 2:397-404 (1992); R6der et al., Mol. Gen. Genet.

243:32-38 (1994), each of which is incorporated herein by reference); ecdysone inducible element (Christopherson et al., Proc. Natl. Acad. Sci. USA 89:6314-6318 (1992), which is incorporated herein by reference); or heat shock inducible element (Takahashi et al., Plant Physiol.

99:383-390 (1992), which is incorporated herein by reference). Another useful inducible regulatory element can be a lac operon element, which is used in combination with a constitutively expressed lac repressor to confer, for example, IPTG-inducible expression, as described by Wilde et al., (EMBO J. 11:1251-1259 (1992), which is incorporated herein by reference).

An inducible regulatory element useful in a method of the invention also can be, for example, a nitrate-inducible promoter derived from the spinach nitrite reductase gene (Back et al., Plant Mol.

WO 97/46079 PCTIUS97/09682 41 Biol. 17:9 (1991), which is incorporated herein by reference) or a light-inducible promoter, such as that associated with the small subunit of RuBP carboxylase or the LHCP gene families (Feinbaum et al., Mol. Gen. Genet.

226:449 (1991); Lam and Chua, Science 248:471 (1990), each of which is incorporated herein by reference). An inducible regulatory element useful in constructing a transgenic seed plant also can be a salicylic acid inducible element (Uknes et al., Plant Cell 5:159-169 (1993); Bi et al., Plant J. 8:235-245 (1995), each of which is incorporated herein by reference) or a plant hormone-inducible element (Yamaguchi-Shinozaki et al., Plant Mol. Biol. 15:905 (1990); Kares et al., Plant Mol.

Biol. 15:225 (1990), each of which is incorporated herein by reference). A human glucocorticoid response element also is an inducible regulatory element that can confer hormone-dependent gene expression in seed plants (Schena et al., Proc. Nat., Acad. Sci. USA 88:10421 (1991), which is incorporated herein by reference).

An inducible regulatory element that is particularly useful for increasing expression of a floral meristem identity gene product in a transgenic seed plant of the invention is a copper inducible regulatory element (see, for example, Mett et al., supra, 1993). Thus, the invention provides a recombinant nucleic acid molecule comprising a copper inducible regulatory element operably linked to a nucleic acid molecule encoding a floral meristem identity gene product and a transgenic seed plant containing such a recombinant nucleic acid molecule.- Copper, which is a natural part of the nutrient environment of a seed plant, can be used to increase expression of a nucleic acid molecule encoding a floral meristem identity gene product operably linked to a copper inducible regulatory element. For example, an ACE1 binding site in conjunction with constitutively expressed yeast ACE1 protein confers copper inducible WO 97/46079 PCT/US97/09682 42 expression upon an operably linked nucleic acid molecule.

The ACE1 protein, a metalloresponsive transcription factor, is activated by copper or silver ions, resulting in increased expression of a nucleic acid molecule operably linked to an ACE1 element.

Such a copper inducible regulatory element can be an ACE1 binding site from the metallothionein gene promoter (SEQ ID NO: 21; Furst et al., Cell 55:705-717 (1988), which is incorporated herein by reference). For example, the ACE1 binding site can be combined with the base-pair domain A of the cauliflower mosaic virus promoter and operably linked to a nucleic acid molecule encoding AP1, CAL or LFY to produce a recombinant nucleic acid molecule of the invention. In a transgenic seed plant constitutively expressing ACE1 under control of such a modified CaMV 35S promoter, for example, copper inducible expression is conferred upon an operably linked nucleic acid molecule encoding a floral meristem identity gene product.

The expression of a nucleic acid encoding a floral meristem identity gene product operably linked to a copper inducible regulatory element, such as 5'-AGCTTAGCGATGCGTCTTTTCCGCTGAACCGTTCCAGCAAAAAAGACTAG-3' (SEQ ID NO: 21), can be increased in a transgenic seed plant grown under copper ion-depleted conditions, for example, and contacted with 50 pM copper sulfate in a nutrient solution or with 0.5 AM copper sulfate applied by foliar spraying of the transgenic seed plant (see, for example, Mett et al., supra, 1993). A single application of 0.5 pM copper sulfate can be sufficient to sustain increased floral meristem identity gene product expression over a period of several days. If desired, a transgenic seed plant of the invention also can be contacted with multiple applications of an inducing agent such as copper sulfate.

WO 97/46079 PCT/US97/09682 43 An inducible regulatory element also can confer tetracycline-dependent floral meristem identity gene expression in a transgenic seed plant of the invention.

Thus, the present invention provides a recombinant nucleic acid molecule comprising a tetracycline inducible regulatory element operably linked to a nucleic acid molecule encoding a floral meristem identity gene product as well as a transgenic seed plant into which such a recombinant nucleic acid molecule has been introduced.

A tetracycline inducible regulatory element is particularly useful for conferring tightly regulated gene expression as indicated by the observation that a phenotype that results from even low amounts of a gene product expression is suppressed from such an inducible system in the absence of inducing agent (see, for example, R6der et al., supra, 1994).

A transgenic seed plant constitutively expressing TnlO-encoded Tet repressor (TetR), for example, can be contacted with tetracyline to increase expression of a nucleic acid molecule encoding a floral meristem identity gene product operably linked to the cauliflower mosaic virus promoter containing several tet operator sequences (5'-ACTCTATCAGTGATAGAGT-3'; SEQ ID NO: 22) positioned close to the TATA box (see, for example, Gatz, Meth. Cell Biol. 50:411-424 (1995), which is incorporated herein by reference; Gatz et al., supra, 1992). Such a tetracycline-inducible system can increase expression of an operably linked nucleic acid molecule as much as 200 to 500-fold in a transgenic angiosperm or gymnosperm of the invention.

A high level of Tet repressor expression (about 1 x 106 molecules per cell) is critical for tight regulation. Thus, a seed plant preferably is transformed first with a plasmid encoding the Tet repressor, and WO 97/46079 PCT/US97/09682 44 screened for high level expression. For example, plasmid pBinTet (Gatz, supra, 1995) contains the Tet repressor coding region, which is expressed under control of the CaMV 35S promoter, and the neomycin phosphotransferase gene for selection of transformants. To screen transformants for a high level of Tet repressor expression, a plasmid containing a reporter gene under control of a promoter with tet operators, such as pTX-Gus-int (Gatz, supra, 1995), can be transiently introduced into a seed plant cell and assayed for activity in the presence and absence of tetracycline.

High -glucouronidase (GUS) expression that is dependent on the presence of tetracycline is indicative of high Tet repressor expression.

A particularly useful tetracycline inducible regulatory element is present in plasmid pBIN-HygTX, which has a CaMV 35S promoter, into which three tet operator sites have been inserted, and an octopine synthase polyadenylation site (Gatz, supra, 1995). A multiple cloning site between the promoter and polyadenylation signal in pBIN-HygTX allows for convenient insertion of a nucleic acid molecule encoding the desired floral meristem identity gene product, and the hygromycin phosphotransferase gene allows for selection of transformants containing the construct. In a preferred embodiment of the invention, previously selected Tet repressor positive cells are transformed with a plasmid such as pBIN-HygTX, into which a nucleic acid molecule encoding a floral meristem identity gene product has been inserted.

To increase floral meristem identity gene product expression using a tetracycline-inducible regulatory element, a transgenic seed plant of the invention can be contacted with tetracycline or, WO 97/46079 PCT/US97/09682 preferably, with chlor-tetracycline (SIGMA), which is a more efficient inducer than tetracycline. In addition, a useful inducing agent can be a tetracycline analog that binds the Tet repressor to function as an inducer but that does not act as an antibiotic (Gatz, supra, 1995).

A transgenic seed plant of the invention can be contacted, for example, by watering with about 1 mg/liter chlor-tetracycline or tetracycline. Similarly, a plant grown in hydroponic culture can be contacted with a solution containing about 1 mg/liter chlor-tetracycline or tetracycline (Gatz, supra, 1995). If desired, a transgenic angiosperm or gymnosperm can be contacted repeatedly with chlor-tetracycline or tetracycline every other day for about 10 days (R6der et al., supra, 1994).

Floral meristem identity gene product expression is increased efficiently at a tetracycline concentration that does not inhibit the growth of bacteria, indicating that the use of tetracycline as an inducing agent will not present environmental concerns.

An ecdysone inducible regulatory element also can be useful in practicing the methods of the invention.

For example, an ecdysone inducible regulatory element can contain four copies of an ecdysone response element having the sequence 5'-GATCCGACAAGGGTTCAATGCACTTGTCA-3' (EcRE; SEQ ID NO: 23) as described in Christopherson et al., supra, 1992. In a transgenic seed plant into which a nucleic acid encoding an ecdysone receptor has been introduced, an ecdysone inducible regulatory element can confer ecdysone-dependent expression on a nucleic acid molecule encoding a floral meristem identity gene product. An appropriate inducing agent for increasing expression of a nucleic acid molecule operably linked to an ecdysone inducible regulatory element can be, for example, a-ecdysone, 20-hydroxyecdysone, polypodine B, ponasterone A, muristerone A or RH-5992, which is an WO 97/46079 PCT/US97/09682 46 ecdysone agonist that mimics 20-hydroxyecdysone (see, for example, Kreutzweiser et al., Ecotoxicol. Environ. Safety 28:14-24 (1994), which is incorporated herein by reference and Christopherson et al., supra, 1992).

Methods for determining an appropriate inducing agent for use with an ecdysone inducible regulatory element are well known in the art. As disclosed herein, compound RH-5992 can be a particularly useful inducing agent for increasing floral meristem gene product expression in a transgenic seed plant containing an ecdysone inducible regulatory element.

An inducible regulatory element also can be derived from the promoter of a heat shock gene, such as HSP81-1 (SEQ ID NO: 24; Takahashi, supra, 1992). Thus, the invention also provides a recombinant nucleic acid molecule comprising a heat shock inducible regulatory element operably linked to a nucleic acid molecule encoding a floral meristem identity gene product and a transgenic seed plant containing such a recombinant nucleic acid molecule. The HSP81-1 promoter (SEQ ID NO: 24) confers low level expression upon an operably linked nucleic acid molecule in parts of roots under unstressed conditions and confers high level expression in most Arabidopsis tissues following heat shock (see, for example, Yabe et al., Plant Cell Physiol.

35:1207-1219 (1994), which is incorporated herein by reference). After growth of Arabidopsis at 23 0 C, a single heat shock treatment at 37 0 C for two hours is sufficient to induce expression of a nucleic acid molecule operably linked to the HSP81-1 gene regulatory element (see Ueda et al., Mol. Gen. Genet. 250:533-539 (1996), which is incorporated herein by reference).

The use of a heat shock inducible regulatory element is particularly useful for a transgenic seed WO 97/46079 PCT/US97109682 47 plant of the invention grown in an enclosed environment such as a green house, where temperature can be readily manipulated. The use of a heat shock inducible regulatory element especially is applicable to a transplantable or potted transgenic seed plant of the invention, which can be moved conveniently from an environment having a low temperature to an environment having a high temperature. A transgenic angiosperm or gymnosperm of the invention containing a recombinant nucleic acid molecule comprising a HSP81-1 heat shock regulatory element operably linked to a nucleic acid molecule encoding a floral meristem identity gene product also can be induced, for example, by altering the ambient temperature, watering with heated water or submersing the transgenic seed plant in a sealed plastic bag into a heated water bath (see, for example, Ueda et al., supra, 1996) A recombinant nucleic acid molecule of the invention comprising an inducible gene regulatory element can be expressed variably in different lines of transgenic seed plants. In some transgenic lines, for example, leaky expression of the introduced recombinant nucleic acid molecule can occur in the absence of the appropriate inducing agent due to phenomena such as position effects (see, for example, Ueda et al., supra, 1996). Thus, a transgenic seed plant containing, a recombinant nucleic acid molecule comprising an inducible gene regulatory element operably linked to a nucleic acid encoding a floral meristem identity gene product can be screened, if desired, to obtain a particular transgenic seed plant in which expression of the operably linked nucleic acid molecule is desirably low in the absence of the appropriate inducing agent.

The present invention also provides a method of converting shoot meristem to floral meristem in an angiosperm by introducing into the angiosperm a recombinant nucleic acid molecule comprising an inducible regulatory element operably linked to a nucleic acid molecule encoding CAL to produce a transgenic angiosperm, and contacting the transgenic angiosperm with an inducing agent, thereby increasing expression of the floral meristem identity gene product and converting shoot meristem to floral meristem in the transgenic angiosperm. In such a method of the invention, the inducible regulatory element can be, for example, a copper inducible element, tetracycline inducible element, ecdysone inducible element or heat shock inducible element.

In addition, the invention provides a method of promoting early reproductive development in a seed plant such as an angiosperm or gymnosperm by introducing into the seed plant a recombinant nucleic acid molecule comprising an inducible regulatory element operably linked to a nucleic acid molecule encoding CAL to produce a transgenic seed plant, and contacting the transgenic seed plant with an inducing agent, thereby increasing expression of the floral meristem identity gene product and promoting early 25 reproductive development in the transgenic seed plant.

In a method of the invention for promoting early reproductive development in a seed plant, the inducible regulatory element can be, for example, a copper inducible element, tetracycline inducible element, 30 ecdysone inducible element or heat shock inducible element eose a oo* The term "inducing agent," as used herein, means a substance or condition that effects increased -sl-exoression of a nucleic acid molecule operably linked to WO 97/46079 PCT/US97/09682 49 a particular inducible regulatory element as compared to the level of expression of the nucleic acid molecule in the absence of the inducing agent. An inducing agent can be, for example, a naturally occurring or synthetic chemical or biological molecule such as a simple or complex organic molecule, a peptide, a protein or an oligonucleotide that increases expression of a nucleic acid molecule operably linked to a particular inducible regulatory element. An example of such an inducing agent is a compound such as copper sulfate, tetracycline or an ecdysone. An inducing agent also can be a condition such as heat of a certain temperature or light of a certain wavelength. When used in reference to a particular inducible regulatory element, an "appropriate" inducing agent means an inducing agent that results in increased expression of a nucleic acid molecule operably linked to the particular inducible regulatory element.

An inducing agent of the invention can be used alone or in solution or can be used in conjunction with an acceptable carrier that can serve to stabilize the inducing agent or to promote absorption of the inducing agent by a seed plant. If desired, a transgenic seed plant of the invention can be contacted with an inducing agent in combination with an unrelated substance such as a plant nutrient, pesticide or insecticide.

One skilled in the art can readily determine the optimum concentration of an inducing agent needed to produce increased expression of a nucleic acid molecule operably linked to an inducible regulatory element in a transgenic seed plant of the invention. For conveniently determining the optimum concentration of inducing agent from a range of useful concentrations, one skilled in the art can operably link the particular inducible regulatory element to a nucleic acid molecule encoding a reporter gene product such as g-glucouronidase (GUS) and assay for WO 97/46079 PCT/US97/09682 reporter gene product activity in the presence of various concentrations of inducing agent (see, for example, Jefferson et al., EMBO J. 6:3901-3907 (1987), which is incorporated herein by reference) As used herein, the term "contacting," in reference to a transgenic seed plant of the invention, means exposing the transgenic seed plant to an inducing agent, or to a cognate ligand as disclosed below, such that the agent can induce expression of a nucleic acid molecule operably linked to the particular inducible regulatory element. A transgenic seed plant such as an angiosperm or gymnosperm, which contains a recombinant nucleic acid molecule of the invention, can be contacted with an inducing agent in a variety of manners.

Expression of a floral meristem identity gene product can be increased conveniently, for example, by spraying a transgenic seed plant with an aqueous solution containing an appropriate inducing agent or by adding an appropriate inducing agent to the water supply of a transgenic seed plant grown using irrigation or to the water supply of a transgenic seed plant grown hydroponically. A transgenic seed plant containing a recombinant nucleic acid molecule of the invention also can be contacted by spraying the seed plant with an inducing agent in aerosol form. In addition, a transgenic seed plant can be contacted with an appropriate inducing agent by adding the agent to the soil or other solid nutrient media in which the seed plant is grown, whereby the inducing agent is absorbed into the seed plant. Other modes of contacting a transgenic seed plant with an inducing agent, such as injecting or immersing the seed plant in a solution containing an inducing agent, are well known in the art.

For an inducing agent that is temperature or light, for example, contacting can be effected by altering the temperature or light to which the transgenic seed plant is exposed, or, if desired, by moving the transgenic seed WO 97/46079 PCTIUS97/09682 51 plant from an environment of one temperature or light source to an environment having the appropriate inducing temperature or light source.

If desired, a transgenic seed plant of the invention can be contacted individually with an inducing agent. Furthermore, a group of transgenic seed plants that, for example, are located together in a garden plot, hot house or field, can be contacted en masse with an inducing agent, such that floral meristem identity gene product expression is increased coordinately in all transgenic seed plants of the group.

A transgenic seed plant of the invention can be contacted with an inducing agent using one of several means. For example, a transgenic seed plant can be contacted with an inducing agent by non-automated means such as with a hand held spraying apparatus. Such manual means can be useful when the methods of the invention are applied to particularly delicate or valuable seed plant varieties or when it is desirable, for example, to promote early reproductive development in a particular transgenic seed plant without promoting early reproductive development in a neighboring transgenic seed plant. Furthermore, a transgenic seed plant of the invention can be contacted with an inducing agent by mechanical means such as with a conventional yard "sprinkler" for a transgenic seed plant grown, for example, in a garden; a mechanical spraying system in a green house; traditional farm machinery for spraying field crops; or "crop dusting" for conveniently contacting an entire field of transgenic seed plants with a particulate or gaseous inducing agent. The skilled practitioner, whether home gardener or commercial farmer, recognizes that these and other manual or mechanical means can be used to contact a transgenic seed plant with WO 97/46079 PCT/US97/09682 52 an inducing agent according to the methods of the invention.

Furthermore, it is recognized that a transgenic seed plant of the invention can be contacted with a single treatment of an inducing agent or, if desired, can be contacted with multiple applications of the inducing agent. In a preferred embodiment of the invention, a transgenic seed plant of the invention is contacted once with an inducing agent to effectively increase floral meristem identity gene product expression, thereby promoting early reproductive development in the transgenic seed plant. Similarly, a transgenic angiosperm of the invention preferably is contacted once with an inducing agent to effectively increase floral meristem identity gene product expression and convert shoot meristem to floral meristem in the transgenic angiosperm.

A single application of an inducing agent is preferable when a transient increase in floral meristem identity gene product expression from a recombinant nucleic acid molecule of the invention promotes irreversible early reproductive development in a seed plant. In many seed plant species, early reproductive development is irreversible. Transient expression of a floral meristem identity gene product from an introduced recombinant nucleic acid molecule, for example, results in sustained ectopic expression of endogenous floral meristem identity gene products, resulting in irreversible early reproductive development. For example, ectopic expression of AP1 in a transgenic plant induces endogenous LFY gene expression, and ectopic expression of LFY induces endogenous AP1 gene expression (Mandel and Yanofsky, Nature 377:522-524 (1995), which is incorporated herein by reference; Weigel and Nilsson, supra, 1995). Genetic studies also indicate that CAL can

LI

n WO 97/46079 PCT/US97/09682 53 act directly or indirectly to increase expression of API and LFY. Thus, ectopic expression of CAL from an exogenous nucleic acid molecule, for example, can induce endogenous API and LFY expression (see Bowman et al., supra, 1993). Enhanced expression of endogenous API, LFY or CAL following a transient increase in expression of an introduced floral meristem identity gene product induced by a single application of an inducing agent can make repeated applications of an inducing agent unnecessary.

In some seed plants, however, such as angiosperms characterized by the phenomenon of floral reversion, repeated applications of the inducing agent can be desirable. In species such as impatiens, an initiated flower can revert into a shoot such that the center of the developing flower behaves as an indeterminate shoot (see, for example, Battey and Lyndon, Ann. Bot. 61:9-16 (1988), which is incorporated by reference herein). Thus, to prevent floral reversion in species such as impatiens, repeated applications of an inducing agent can be useful. Repeated applications of an inducing agent, as well as single applications, are encompassed within the scope of the present invention.

The invention further provides a nucleic acid molecule encoding a chimeric protein, which comprises a nucleic acid molecule encoding a floral meristem identity gene product such as API, CAL or LFY linked in frame to a nucleic acid molecule encoding a ligand binding domain.

Expression of a chimeric protein of the invention in a seed plant is useful because the ligand binding domain renders the activity of a linked gene product dependent on the presence of cognate ligand. Specifically, in a chimeric protein of the invention, floral meristem gene product activity is increased in the presence of cognate ligand, as compared to activity in the absence of cognate ligand.

/I

r WO 97/46079 PCT/US97/09682 54 A nucleic acid molecule encoding a chimeric protein of the invention comprises a nucleic acid molecule encoding a floral meristem identity gene product, such as a nucleic acid molecule having the nucleic acid sequence SEQ ID NO: 1, SEQ ID NO: 9 or SEQ ID NO: 15, which encodes AP1, CAL or LFY, respectively, any of which is linked in frame.to a nucleic acid molecule encoding a ligand binding domain. The expression of such a nucleic acid molecule results in the production of a chimeric protein containing a floral meristem identity gene product fused to a ligand binding domain. Thus, the invention also provides a chimeric protein containing a floral meristem identity gene product fused to a ligand binding domain and an antibody that specifically binds such a chimeric protein.

The invention further provides a transgenic seed plant, such as angiosperm or gymnosperm, that contains a nucleic acid molecule encoding a chimeric protein of the invention. The invention provides, for example, a transgenic seed plant containing a nucleic acid molecule encoding a chimeric protein, which comprises a nucleic acid molecule encoding AP1, CAL or LFY linked in frame to a nucleic acid molecule encoding a ligand binding domain. A particularly useful transgenic seed plant contains a nucleic acid molecule encoding AP1 linked in frame to a nucleic acid molecule encoding an ecdysone receptor ligand binding domain or a glucocorticoid receptor ligand binding domain. The invention also provides a transgenic seed plant containing a nucleic acid molecule encoding a chimeric protein, which comprises a nucleic acid molecule encoding CAL linked in frame to a nucleic acid molecule encoding an ecdysone receptor ligand binding domain or a glucocorticoid receptor ligand binding domain. In addition, there is provided a transgenic seed plant

J

WO 97/46079 PCT/US97/09682 containing a nucleic acid molecule encoding a chimeric protein, which comprises a nucleic acid molecule encoding LFY linked in frame to a nucleic acid molecule encoding an ecdysone receptor ligand binding domain or a glucocorticoid receptor ligand binding domain.

Any floral meristem identity gene product, as defined herein, is useful in a chimeric protein of the invention. Thus, a nucleic acid molecule encoding Arabidopsis thaliana AP1 (SEQ ID NO: Brassica oleracea AP1 (SEQ ID NO: Brassica oleracea var.

Botrytis AP1 (SEQ ID NO: 8) or Zea mays AP1 (SEQ ID NO: 10), each of which have activity in converting shoot meristem to floral meristem, can be used to construct a nucleic acid molecule encoding a chimeric protein of the invention. Similarly, a nucleic acid molecule encoding, for example, Arabidopsis thaliana CAL (SEQ ID NO: Brassica oleracea CAL (SEQ ID NO: 12), or a nucleic acid molecule encoding Arabidopsis thaliana LFY (SEQ ID NO: 16) is useful when linked in frame to a nucleic acid molecule encoding a ligand binding domain to produce a nucleic acid molecule encoding a ligand-dependent chimeric protein of the invention.

A ligand binding domain useful in a chimeric protein of the invention is a domain that, when fused in frame to a heterologous gene product, renders the activity of the fused gene product dependent on cognate ligand such that the activity of the fused gene product is increased in the presence of cognate ligand as compared to its activity in the absence of ligand. Such a ligand binding domain can be a steroid binding domain such as the ligand binding domain of an ecdysone receptor, glucocorticoid receptor, estrogen receptor, progesterone receptor, androgen receptor, thyroid receptor, vitamin D receptor or retinoic acid receptor.

,l' WO 97/46079 PCT/US97/09682 56 A particularly useful ligand binding domain is the ecdysone receptor ligand binding domain contained within amino acids 329 to 878 of the Drosophila ecdysone receptor (SEQ ID NO: 18); Koelle et al., Cell 67:59-77 (1991); Thummel, Cell 83:871-877 (1995), each of which is incorporated herein by reference) or a glucocorticoid receptor ligand binding domain, encompassed, for example, within amino acids 512 to 795 of the rat glucocorticoid receptor (SEQ ID NO: 20; Miesfeld et al., Cell 46:389-399 (1986), which is incorporated herein by reference).

A chimeric protein of the invention containing an ecdysone receptor ligand binding domain has floral meristem identity gene product activity that can be increased in the presence of ecdysone ligand. Similarly, a chimeric protein of the invention containing a glucocorticoid receptor ligand binding domain has floral meristem identity gene product activity that is increased in the presence of glucocorticoid ligand. It is well known that in a chimeric protein containing a heterologous gene product such as adenovirus E1A, c-myc, c-fos, the HIV-1 Rev transactivator, MyoD or maize regulatory factor R fused to the rat glucocorticoid receptor ligand binding domain, activity of the fused heterologous gene product can be increased by glucocorticoid ligand (Eilers et al., Nature 340:66 (1989); Superti-Furga et al., Proc. Natl. Acad. Sci..

U.S.A. 88:5114 (1991); Hope et al., Proc. Natl. Acad.

Sci.. U.S.A. 87:7787 (1990); Hollenberg et al., Proc.

Natl. Acad. Sci.. U.S.A. 90:8028 (1993), each of which is incorporated herein by reference).

A nucleic acid molecule encoding a chimeric protein of the invention can be introduced into a seed plant where, under appropriate conditions, the chimeric protein is expressed. In such a transgenic seed plant, floral meristem identity gene product activity can be WO 97/46079 PCT/US97/09682 57 increased by contacting the transgenic seed plant with cognate ligand. For example, activity of a heterologous protein fused to a rat glucocorticoid receptor ligand binding domain (amino acids 512 to 795) expressed under the control of the constitutive cauliflower mosaic virus promoter in Arabidopsis was low in the absence of glucocorticoid ligand; whereas, upon contacting the transformed plants with a synthetic glucocorticoid, dexamethasone, activity of the protein was increased greatly (Lloyd et al., Science 266:436-439 (1994), which is incorporated herein by reference). As disclosed herein, a ligand binding domain fused to a floral meristem identity gene product renders the activity of a fused floral meristem identity gene product ligand-dependent such that, upon contacting the transgenic seed plant with cognate ligand, floral meristem identity gene product activity is increased.

Methods for constructing a nucleic acid molecule encoding a chimeric protein of the invention are routine and well known in the art (Sambrook et al., supra, 1989). Methods of constructing, for example, a nucleic acid encoding an AP1-glucocorticoid receptor ligand binding domain chimeric protein are described in Example IV. For example, the skilled artisan recognizes that a stop codon encoded by the nucleic acid molecule must be removed and that the two nucleic acid molecules must be linked in frame such that the reading frame of the 3' nucleic acid molecule coding sequence is preserved. Methods of transforming a seed plant such as an angiosperm or gymnosperm with a nucleic acid molecule are disclosed above and well known in the art (see Examples I, II and III; see, also, Mohoney et al., U.S.

patent number 5,463,174, and Barry et al., U.S. patent number 5,463,175, each of which is incorporated herein by reference).

WO 97/46079 PCT/US97/09682 58 As used herein, the term "linked in frame," when used in reference to two nucleic acid molecules that make up a nucleic acid molecule encoding a chimeric protein, means that the two nucleic acid molecules are linked in the correct reading frame such that, under appropriate conditions, a full-length chimeric protein is expressed. In particular, a 5' nucleic acid molecule, which encodes the amino-terminal portion of the chimeric protein, must be linked to a 3' nucleic acid molecule, which encodes the carboxyl-terminal portion of the chimeric protein, such that the carboxyl-terminal portion of the chimeric protein is translated in the correct reading frame. One skilled in the art would recognize that a nucleic acid molecule encoding a chimeric protein of the invention can comprise, for example, a 5' nucleic acid molecule encoding a floral meristem identity gene product linked in frame to a 3' nucleic acid molecule encoding a ligand binding domain or can comprise a nucleic acid molecule encoding a ligand binding domain linked in frame to a 3' nucleic acid molecule encoding a floral meristem identity gene product. Preferably, a nucleic acid molecule encoding a chimeric protein of the invention comprises a 5' nucleic acid molecule encoding a floral meristem identity gene product linked in frame to a 3' nucleic acid molecule encoding a ligand binding domain.

In a transgenic angiosperm containing a chimeric protein of the invention, conversion of shoot meristem to floral meristem can be induced by contacting the transgenic angiosperm with a cognate ligand that is absorbed by the angiosperm and binds the chimeric protein within its ligand binding domain. Thus, the present invention provides a method of converting shoot meristem to floral meristem in an angiosperm by introducing into the angiosperm a nucleic acid molecule encoding a chimeric protein to produce a transgenic angiosperm, WO 97/46079 PCT/US97/09682 59 where, under appropriate conditions, the chimeric protein containing a floral meristem identity gene product fused to a ligand binding domain is expressed; and contacting the transgenic angiosperm with cognate ligand, where, upon binding of the cognate ligand to the ligand binding domain, floral meristem identity gene product activity is increased, thereby converting shoot meristem to floral meristem in the transgenic angiosperm.

The present invention provides, for example, a method of converting shoot meristem to floral meristem in an angiosperm by introducing into the angiosperm a nucleic acid molecule encoding a chimeric protein, which comprises a nucleic acid molecule encoding API, CAL or LFY linked in frame to a nucleic acid molecule encoding an ecdysone receptor ligand binding domain, to produce a transgenic angiosperm, where, under appropriate conditions, the chimeric protein is expressed; and contacting the transgenic angiosperm with ecdysone ligand, where, upon binding of the ecdysone ligand to the ecdysone receptor ligand binding domain, floral meristem identity gene product activity is increased, thereby converting shoot meristem to floral meristem in the transgenic angiosperm. Similarly, the invention provides, for example, a method of converting shoot meristem to floral meristem in an angiosperm by introducing into the angiosperm a nucleic acid molecule encoding a chimeric protein, which comprises a nucleic acid molecule encoding AP1, CAL or LFY linked in frame to a nucleic acid molecule encoding a glucocorticoid receptor ligand binding domain, to produce a transgenic angiosperm, where, under appropriate conditions, the chimeric protein is expressed; and contacting the transgenic angiosperm with glucocorticoid ligand, where, upon binding of the glucocorticoid ligand to the glucocorticoid receptor ligand binding domain, floral meristem identity gene product activity is increased, WO 97/46079 PCT/US97/09682 thereby converting shoot meristem to floral meristem in the transgenic angiosperm.

In addition, the invention provides a method of promoting early reproductive development in a seed plant by introducing into the seed plant a nucleic acid molecule encoding a chimeric protein of the invention to produce a transgenic seed plant, where, under appropriate conditions, the chimeric protein containing a floral meristem identity gene product fused to a ligand binding domain is expressed; and contacting the transgenic seed plant with cognate ligand, where, upon binding of the cognate ligand to the ligand binding domain, floral meristem identity gene product activity is increased, thereby promoting early reproductive development in the transgenic seed plant. The methods of the invention can be practiced with numerous seed plant varieties. The seed plant can be, for example, an angiosperm such as a cereal plant, leguminous plant, hardwood tree or coffee plant, or can be a gymnosperm such as a pine, fir, spruce or redwood tree.

There is provided, for example, a method of promoting early reproductive development in a seed plant by introducing into the seed plant a nucleic acid molecule encoding a chimeric protein, which comprises a nucleic acid molecule encoding a floral meristem identity gene product linked in frame to a nucleic acid molecule encoding an ecdysone receptor ligand binding domain, to produce a transgenic seed plant, where, under appropriate conditions, the chimeric protein is expressed; and contacting the transgenic seed plant with ecdysone ligand, where, upon binding of the ecdysone ligand to the ecdysone receptor ligand binding domain, floral meristem identity gene product activity is increased, thereby promoting early reproductive development in the transgenic seed plant. Similarly, the invention 4 WO 97/46079 PCT/US97/09682 61 provides, for example, a method of promoting early reproductive development in a seed plant by introducing into the seed plant a nucleic acid molecule encoding a chimeric protein, which comprises a nucleic acid molecule encoding AP1, CAL or LFY linked in frame to a nucleic acid molecule encoding a glucocorticoid receptor ligand binding domain, to produce a transgenic seed plant, where, under appropriate conditions, the chimeric protein is expressed; and contacting the transgenic seed plant with glucocorticoid ligand, where, upon binding of the glucocorticoid ligand to the glucocorticoid receptor ligand binding domain, floral meristem identity gene product activity is increased, thereby promoting early reproductive development in the transgenic seed plant.

As used herein, the term "ligand" means a naturally occurring or synthetic chemical or biological molecule such as a simple or complex organic molecule, a peptide, a protein or an oligonucleotide that specifically binds a ligand binding domain. In the methods of the present invention, a ligand can be used alone or in solution or can be used in conjunction with an acceptable carrier that can serve to stabilize the ligand or promote absorption of the ligand by a seed plant. If desired, a transgenic seed plant of the invention can be contacted with a ligand for increasing floral meristem identity gene product activity in combination with an unrelated molecule such as a plant nutrient, pesticide or insecticide. When used in reference to a particular ligand binding domain, the term "cognate ligand" means a ligand that, under suitable conditions, specifically binds the particular ligand binding domain.

One skilled in the art readily can determine the optimum concentration of cognate ligand needed to bind a ligand binding domain and increase floral meristem WO 97/46079 PCT/US97/09682 62 identity gene product activity in a transgenic seed plant of the invention. Generally, a concentration of about 1 nM to 10 pM cognate ligand is useful for increasing floral meristem identity gene product activity in a transgenic seed plant expressing a chimeric protein of the invention. Preferably, a concentration of about 100 nM to 1 /M cognate ligand is useful for increasing floral meristem identity gene product activity in a transgenic seed plant containing a chimeric protein of the invention (see, for example, Christopherson et al., Proc. Natl.

Acad. Sci. USA 89:6314-6318 (1992), which is incorporated herein by reference; also, see Lloyd et al., supra, 1994). For example, a concentration of about 100 nM to 1 AM dexamethasone can be useful for increasing floral meristem identity gene product activity in a transgenic seed plant of the invention containing a nucleic acid molecule encoding a chimeric protein, which comprises a nucleic acid molecule encoding a floral meristem identity gene product, such as AP1 or CAL, linked in frame to a nucleic acid molecule encoding a glucocorticoid receptor ligand binding domain, as described in Example IV.

As discussed above, a transgenic seed plant of the invention, such as a transgenic seed plant expressing a chimeric protein of the invention, can be contacted in a variety of manners. A transgenic seed plant can be contacted with cognate ligand, for example, by spraying the seed plant with a gaseous ligand or with solution such as an aqueous solution containing the appropriate ligand; or by adding the cognate ligand to the water supply of a seed plant grown using irrigation or grown hydroponically; or by adding the cognate ligand to the soil or other solid nutrient medium in which a seed plant is grown, whereby the cognate ligand is absorbed into the seed plant to increase floral meristem identity gene product activity. A transgenic seed plant expressing a chimeric protein of the invention also can be contacted WO 97/46079 PCT/US97/09682 63 with a cognate ligand in aerosol form. In addition, a transgenic seed plant can be contacted with cognate ligand by injecting the seed plant or by immersing the seed plant in a solution containing the cognate ligand.

A transgenic seed plant expressing a chimeric protein of the invention can be contacted individually with cognate ligand, or a group of transgenic seed plants can be contacted en masse to increase floral meristem gene product activity synchronously in all seed plants of the group. Furthermore, a variety of means can be used to contact a transgenic seed plant of the invention with cognate ligand to increase floral meristem identity gene product activity. A transgenic seed plant can be contacted with cognate ligand using, for example, a hand held spraying apparatus; conventional yard "sprinkler"; mechanical spraying system, such as an overhead spraying system in a green house; traditional farm machinery, or "crop dusting." As discussed above in regard to the application of inducing agents, the methods of the invention can be practiced using these and other manual or mechanical means to contact a transgenic seed plant with single or multiple applications of cognate ligand.

The nucleic acid molecules encoding floral meristem identity gene products provided herein also can be useful in generating sterile transgenic seed plants and in methods of producing reproductive sterility in seed plants. The methods of the invention involve cosuppression metholodology, where a nucleic acid molecule in the sense orientation is introduced into a seed plant to suppress expression of a homologous endogenous gene, or involve antisense metholodology.

Thus, the present invention provides cosuppression and antisense methods of producing reproductively sterile transgenic seed plants as well as the two types of sterile transgenic seed plants produced by these methods.

WO 97/46079 PCTIUS97/09682 64 A method of the invention for producing a reproductively sterile transgenic seed plant has a variety of uses including safely growing transgenic trees in close contact with interfertile wild trees, increasing wood production and reducing allergenic pollen production. A method for producing reproductive sterility in seed plants, which is useful for transgene containment, can allow, for example, the introduction of transgenic trees into the environment. Of particular concern to the introduction of transgenic trees into the environment is the possibility of enhanced "weediness" or the movement of transgenes by cross-fertilization into gene pools of wild relatives. Most commercially grown forest trees, for example, are grown in close proximity to interfertile wild populations, and gene flow within and among tree populations usually is extensive, making the probability of transgene escape from plantations of fertile transgenic trees high.. Regulatory agencies have based approval of transgenic tree planting on sexual isolation of the transgenic species; for example, approval of two field tests for transgenic poplars by the Animal and Plant Health Inspection Service (APHIS) was contingent on the trees not being allowed to flower (see, for example, Strauss et al., Molec. Breed 1:5-26 (1995), which is incorporated herein by reference). Thus, transgene containment through, for example, the use of sterile transgenic trees is central to the usefulness of improved transgenic varieties.

Methods of producing reproductively sterile seed plants also can be useful for increasing wood production, since substantial energy and nutrients are committed to reproductive development in trees. For example, in trees such as radiata pine, white spruce, balsam fir and Douglas fir, reduced growth, as measured by height or stem volume, is correlated with the early production of cones (Strauss et al., supra, 1995). Thus, WO 97/46079 PCT/US97/09682 the methods of the invention, which prevent flowering or cone development, for example, by producing reproductive sterility, are useful for growing substantially larger trees, thus increasing wood production.

A method for producing reproductively sterile seed plants also can be useful for alleviating allergies caused by tree pollen. For example, in Japan many people suffer from allergies caused by the most commonly planted forest tree, the conifer sugi (Strauss et al., supra, 1995). The methods of the invention, therefore, can be advantageous for preventing pollen formation in seed plants such as the conifer sugi.

Cosuppression, which relies on expression of a nucleic acid molecule in the sense orientation, is a well known methodology that produces coordinate silencing of the introduced nucleic acid molecule and the homologous endogenous gene (see, for example, Flavell, Proc. Natl.

Acad. Sci.. USA 91:3490-3496 (1994), which is incorporated herein by reference; Kooter and Mol, supra, 1993). Although the mechanism of cosuppression is unknown, cosuppression is induced most strongly by a large number of transgene copies or by overexpression of transgene RNA; cosuppression also can be enhanced by modification of the transgene such that it fails to be translated. Cosuppression has been used successfully to produce sterile plants; for example, a sense nucleic acid molecule containing a full-length fbpl coding sequence under control of the strong CaMV 35S promoter has been introduced into petunia. Two of twenty-one transformants exhibited an abnormal phenotype and contained multiple copies of the fbpl transgene. Furthermore, fbpl expression was undetectable in these sterile transgenic plants, indicating that expression of endogenous fbpl was 66 suppressed (Angenent et al., The Plant Journal 4:101-112 (1993), which is incorporated herein by reference).

Antisense nucleic acid molecules, which can act by reducing mRNA translation or by increasing mRNA degradation, for example, also can suppress gene expression of diverse genes and seed plant species (see, for example, Kooter and Mol, Current Opin. Biol.

4:166-171 (1993), which is incorporated herein by reference; see also Strauss et al., supra, 1995).

Antisense nucleic acid molecules previously have been used to successfully suppress the expression of a homologous endogenous gene, thereby generating sterile plants. For example, an antisense chalcone synthase gene under control of the CaMV 35S promoter with an anther-specific enhancer sequence effectively suppressed endogenous chalcone synthase expression levels, resulting in male sterility in transgenic petunia plants (van der Meer et al., The Plant Cell Vol 4:253-262 (1992), which 20 is incorporated herein by reference). Similarly, the full-length tomato TM5 MADS box gene, when placed in antisense orientation under control of the CaMV promoter, was used to produce sterile transgenic tomato plants (Pnuell et al., The Plant Cell Vol. 6, 175-186 (1994), which is incorporated herein by reference).

Antisense nucleic acid molecules encoding floral meristem identity gene products similarly can be used to produce reproductive sterility in seed plants; however, by preventing reproductive development at the earliest 30 stage, the methods of the invention result in an advantageous energy savings.

Also provided is a sterile transgenic seed plant such as an angiosperm or gymnosperm containing one or more sense or antisense nucleic acid molecules encoding a floral meristem identity gene product, or a fragment thereof, such that expression of ,LiP. 1, 1 \I 11 sl- 1--,967 19 07 i O -67 AP1 and LFY gene products, including expression of endogenous API and LFY gene products, is suppressed in the transgenic seed plant. The invention also provides, for example, a sterile transgenic seed plant containing a sense or antisense nucleic acid molecule encoding AP1, or a fragment thereof; a sense or antisense nucleic acid molecule encoding CAL, or a fragment thereof; and a sense or antisense nucleic acid molecule encoding LFY, or a fragment thereof, such that expression of AP1 and LFY gene products, including expression of endogenous AP. and LFY gene products, is suppressed in the transgenic seed plant. The invention further provides a sterile transgenic seed plant containing a sense or antisense nucleic acid molecule encoding AP1, or a fragment thereof, and a sense or antisense nucleic acid molecule encoding LFY, or a fragment thereof, such that expression of AP1 and LFY gene products, including expression of endogenous AP1 and LFY gene products, is suppressed in the transgenic seed plant.

20 Also provided is a method of producing reproductive sterility in a seed plant such as a tree by introducing into a seed plant one or more sense or antisense nucleic acid molecules encoding a floral meristem identity gene product, or a fragment thereof, to produce a transgenic seed plant, such that expression of e API and LFY gene products, including expression of endogenous AP1 and LFY gene products, is suppressed in the transgenic seed plant. In a preferred embodiment of the invention, there are provided methods of producing reproductive sterility in a seed plant by introducing into a seed plant a sense or antisense nucleic acid molecule encoding AP1, or a fragment thereof; a sense or antisense nucleic acid molecule encoding CAL, or a fragment thereof; and a sense or antisense nucleic acid molecule encoding LFY, or a fragment thereof, to produce a transgenic seed plant, such that expression of API and \BP.Is I h, I I I\ 11 11) 1, 68 LFY gene products, including expression of endogenous

API

and LFY gene products, is suppressed in the transgenic seed plant. In another embodiment, the invention provides methods of producing reproductive sterility in a seed plant by introducing into a seed plant a sense or antisense nucleic acid molecule encoding AP1, or a fragment thereof, and a sense or antisense nucleic acid molecule encoding LFY, or a fragment thereof, to produce a transgenic seed plant, such that expression of API and LFY gene products, including expression of endogenous

API

and LFY gene products, is suppressed in the transgenic seed plant.

Sterile seed plants that lack expression of functional API and LFY gene products have been described previously. For example, a non-flowering Arabidopsis Ify apl double mutant has been described in which flowers were transformed into shoot-like structures (see, for example, Bowman et al., supra, 1993, and Weigel, supra, 20 1995). However, in contrast to previously described methods of generating sterile seed plants using mutagenesis, a methodology that is cumbersome or unfeasible in higher plants, the present invention provides a convenient method of producing reproductive sterility in a seed plant using sense or antisense nucleic acid molecules encoding floral meristem identity gene products.

The methods for producing reproductive sterility rely upon introducing into a seed plant one or more sense or antisense nucleic acid molecules encoding a floral meristem identity gene product, or a fragment thereof, such that expression of AP1 and LFY gene products, including expression of endogenous AP1 and LFY gene products, is suppressed in the transgenic seed plant. The skilled artisan will recognize that effective suppression of endogenous

API

BP I I 1-1- 1 K~sp-\2967 10l 91*07 100 WO 97/46079 PCT/US97/09682 69 and LFY gene product expression depends upon the one or more introduced nucleic acid molecules having a high percentage of homology with the corresponding endogenous gene loci.

The homology requirement for effective suppression using sense or antisense nucleic acid molecules can be determined empirically. In general, a minimum of about 80-90% nucleic acid sequence identity is preferred for effective suppression of endogenous floral meristem identity gene product expression. Thus, a nucleic acid molecule encoding a gene ortholog from the family or genus of the seed plant species into which the nucleic acid molecule is to be introduced is preferable in practicing the methods of the invention. More preferably, a nucleic acid molecule encoding a gene ortholog from the same seed plant species into which the nucleic acid molecule is to be introduced is used in the methods of the invention. Although a highly homologous nucleic acid molecule is preferred in the methods of the invention, the sense or antisense nucleic acid molecule need not contain the entire coding sequence of the floral meristem identity gene sequence to be suppressed. Thus, a sense or antisense nucleic acid molecule encoding only a fragment of API, CAL or LFY coding sequence, for example, also can be useful in the methods of the invention.

As used herein in reference to a nucleic acid molecule encoding a floral meristem identity gene product, the terms "sense" and "antisense" have their commonly understood meanings.

As used herein in reference to a nucleic acid molecule encoding a floral meristem identity gene product, the term "fragment" means a portion of the nucleic acid sequence containing at least about 50 base pairs to the full-length of the nucleic acid molecule encoding the floral meristem identity gene product. In contrast to an active fragment, as defined herein, a fragment of a nucleic acid molecule encoding a floral meristem identity gene product need not encode a functional portion of a gene product.

In the methods of the invention for producing reproductive sterility, the sense or antisense nucleic acid molecule is expressed under control of a strong promoter that is expressed, at least in part, in floral meristem. The constitutive cauliflower mosaic virus promoter (Odell et al., supra, 1985), for example, or other strong promoters as disclosed herein, can be useful in the methods of the invention. In addition, an RNA polymerase III promoter can be useful in methods of producing reproductive sterility using an antisense nucleic acid molecule (see, for example, Bourque and Folk, Plant Mol. Biol. 19:641-647 (1992), which is S 20 incorporated herein by reference).

Also provided are novel substantially purified nucleic acid molecules encoding floral meristem identity gene products, in particular a substantially purified nucleic acid molecule encoding Brassica oleracea API having the amino acid sequence SEQ ID NO: 4; a substantially purified nucleic acid molecule encoding Brassica oleracea var. botrytis AP1 having the amino acid sequence SEQ ID NO: 6; or a substantially purified nucleic acid molecule encoding Zea mays AP1 having the amino acid sequence SEQ ID NO: 8. In addition, there is provided a substantially purified nucleic acid molecule that encodes a Brassica oleracea AP1, Brassica oleracea var. botrytis API or Zea mays API and that contains additional 5' or 3' noncoding sequence.

For example, a substantially purified nucleic acid %HP I 1 \huenS\ I -be IH\ ;I-i 3967 1907/00 71 molecule having a nucleotide sequence such as SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 is provided.

As used herein in reference to a particular nucleic acid molecule or gene product, the term "substantially purified" means that the particular nucleic acid molecule or gene product is in a form that is relatively free from contaminating lipids, unrelated gene products, unrelated nucleic acids or other cellular material normally associated with the particular nucleic acid molecule or gene product in a cell.

Also provided is a nucleotide sequence having at least ten contiguous nucleotides of a nucleic acid molecule encoding Brassica oleracea AP1, Brassica oleracea var. botrytis API or Zea mays AP1, provided that said nucleotide sequence is not present in a nucleic acid molecule encoding a MADS domain containing protein. In particular, such a nucleotide sequence can have at least ten contiguous nucleotides of a nucleic acid molecule encoding an AP1 gene product having the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8. A nucleotide sequence of the invention can have, for example, at least ten contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7.

As used herein, the term "contiguous," as used in reference to the nucleotides of a nucleic acid molecule means that the nucleotides of the nucleic acid molecule follow continuously in sequence. Thus, a nucleotide sequence of the invention has at least ten contiguous nucleotides of one of the recited nucleic acid molecules without any extraneous intervening nucleotides.

Explicitly excluded from a nucleotide sequence ,/jq 4 of the present invention is a nucleotide sequence having I A 1 h A.h-i I. 1 00 72 at least ten contiguous nucleotides that is present in a nucleic acid molecule encoding a MADS domain containing protein. MADS domain containing proteins are well known in the art as described in Purugganan et al., supra, S 1995.

In general, a nucleotide sequence of the invention can range in size from about 10 nucleotides to the full-length of a cDNA. Such a nucleotide sequence 1 can be chemically synthesized, using routine methods or can be purchased from a commercial source. In addition, such a nucleotide sequence can be obtained by enzymatic methods such as random priming methods, polymerase chain reaction (PCR) methods or by standard restriction endonuclease digestion, followed by denaturation (Sambrook et al., supra, 1989).

A nucleotide sequence of the invention can be useful, for example, as a primer for PCR (Innis et al.

20 PCR Protocols: A Guide to Methods and Applications, San Diego, CA: Academic Press, Inc. (1990)). Such a nucleotide sequence generally contains from about 10 to about 50 nucleotides.

These nucleotide sequences can be useful in screening a cDNA or genomic library to obtain a related nucleotide sequence. For example, a cDNA library that is prepared from rice or wheat can be screened with a nucleotide sequence having at least ten contiguous nucleotides of the nucleic acid molecule encoding Zea mays API (SEQ ID NO: 7) in order to isolate S" a rice or wheat ortholog of API. Generally, a nucleotide sequence useful for screening a cDNA or genomic library contains at least about 14 to 16 contiguous nucleotides depending, for example, on the hybridization conditions to be used. A nucleotide sequence containing at least 18 SIPRISIlb n I I, I II% i i 3 2 '167.dc 19.0'77/00 73 to 20 nucleotides, or containing at least 21 to nucleotides, also can be useful.

A nucleotide sequence having at least ten contiguous nucleotides of a nucleic acid molecule encoding Zea mays API (SEQ ID NO: 7) also can be used to screen a Zea mays cDNA library to isolate a sequence that is related to but distinct from API. Similarly, a nucleotide sequence having at least ten contiguous nucleotides of a nucleic acid molecule encoding Brassica oleracea AP1 (SEQ ID NO: 3) or a nucleotide sequence having at least ten contiguous nucleotides of a nucleic acid molecule encoding Brassica oleracea var. botrytis AP1 (SEQ ID NO: 5) can be used to screen a Brassica oleracea or Brassica oleracea var. botrytis cDNA library to isolate a novel sequence that is related to but distinct from AP1. In addition, these nucleotide sequences can be useful in analyzing RNA levels or 20 patterns of expression, as by northern blotting or by in situ hybridization to a tissue section. Such a nucleotide sequence also can be used in Southern blot analysis to evaluate gene structure and identify the presence of related gene sequences.

oo ~25 Also provided is a vector containing a nucleic acid molecule encoding a Brassica oleracea AP1 gene product, Brassica oleracea var. botrytis AP1 gene product or Zea mays AP1 gene product. A vector can be a cloning vector or an expression vector and provides a means to transfer an exogenous nucleic acid molecule into a host cell, which can be a prokaryotic or eukaryotic cell. Such vectors are well known and include plasmids, phage vectors and viral vectors. Various vectors and methods for introducing such vectors into a cell are BP 1 1, 1 Ili Sl- i 2 9 llt-' L9. 07,00 74 described, for example, by Sambrook et al., supra, 1989, and by Glick and Thompson, supra, 1993).

Also provided is a-method of producing an AP1 gene product by expressing a nucleic acid molecule encoding an AP1 gene product. Thus, a Brassica oleracea AP1'gene product can be produced according to a method of the invention by expressing a nucleic acid molecule having the amino acid sequence of 1 0 SEQ ID NO: 4 or by expressing a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 3.

Similarly, a Brassica oleracea var. botrytis AP1 gene product can be produced according to a method of the invention by expressing a nucleic acid molecule having the amino acid sequence of SEQ ID NO: 6 or by expressing a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 5. A Zea mays AP1 gene product can be produced by expressing a nucleic acid molecule having the S 20 amino acid sequence of SEQ ID NO: 8 or by expressing a nucleic acid molecule having the nucleic acid sequence of 99 SEQ ID NO: 7.

99 Also provided is a substantially purified AP1 gene product, such as a substantially 25 purified Brassica oleracea API gene product having amino :I acid sequence SEQ ID NO: 4; a substantially purified Brassica oleracea var. botrytis API gene product having amino acid sequence SEQ ID NO: 6; or a substantially purified Zea mays AP1 gene product having amino acid sequence SEQ ID NO: 8. As used herein, the term "gene product" is used in its broadest sense and includes proteins, polypeptides and peptides, which are related in that each consists of a sequence of amino acids joined by peptide bonds. For convenience, the terms "gene product," "protein" and "polypeptide" are used Sinterchangeably. While no specific attempt is made to .UP.IS L hoe51 IIn I H\So c 3 n -I d 9- Ji WO 97/46079 PCT/US97/09682 distinguish the size limitations of a protein and a peptide, one skilled in the art would understand that proteins generally consist of at least about 50 to 100 amino acids and that peptides generally consist of at least two amino acids up to a few dozen amino acids. The term gene product as used herein includes any such amino acid sequence.

An active fragment of a floral meristem identity gene product also can be useful in the methods of the invention. As used herein, the term "active fragment," means a polypeptide portion of a floral meristem identity gene product that can convert shoot meristem to floral meristem in an angiosperm. An active fragment of an AP1 gene product can consist, for example, of an amino acid sequence that is derived from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 and has activity in converting shoot meristem to floral meristem in an angiosperm. An active fragment can be, for example, an amino terminal, carboxyl terminal or internal fragment of Zea mays AP1 (SEQ ID NO: 8) that has activity in converting shoot meristem to floral meristem in an angiosperm. The skilled artisan will recognize that an active fragment of a floral meristem identity gene product, as defined herein, can be useful in the methods of the invention for converting shoot meristem to floral meristem in an angiosperm, for producing early reproductive development in a seed plant, or for producing reproductive sterility in a seed plant.

Such an active fragment can be produced using well known recombinant DNA methods (Sambrook et al., supra, 1989). Similarly, an active fragment can be, for example, an amino terminal, carboxyl terminal or internal fragment of Arabidopsis thaliana CAL (SEQ ID NO: 10) or Brassica oleracea CAL (SEQ ID NO: 12) that has activity, 76 for example, in converting shoot meristem to floral meristem in an angiosperm. The product of the BobCAL gene (SEQ ID NO: 24), which is truncated at amino acid 150, lacks activity in converting shoot meristem to floral meristem and, therefore, is an example of a polypeptide portion of a CAL floral meristem identity gene product that is not an "active fragment" of a floral meristem identity gene product.

An active fragment of a floral meristem identity gene product, which can convert shoot meristem to floral meristem in an angiosperm, can be identified using the methods described in Examples I, II and III.

Briefly, an angiosperm such as Arabidopsis can be transformed with a nucleic acid molecule encoding a portion of a floral meristem identity gene product in order to determine whether the portion can convert shoot meristem to floral meristem and, therefore, is an active fragment of a floral meristem identity gene product.

20 Also provided is an antibody that specifically binds an API gene product having the amino acid sequence of Brassica oleracea AP1 (SEQ ID NO: 4); the amino acid sequence of Brassica oleracea var.

botrytis AP1 (SEQ ID NO: or the amino acid sequence of Zea mays AP1 (SEQ ID NO: As used herein, the term "antibody" is used in its broadest sense to include naturally occurring and non-naturally occurring polyclonal and monoclonal antibodies, as well as a polypeptide fragment of an antibody that retains a .specific binding activity of at least about 1 x 10 S

M-

1 and preferably about 1 x 106 M- 1 for an API gene product having amino acid sequence SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8. One skilled in the art would know that an antibody fragment such as a Fab, or Fv fragment e can retain specific binding activity for an API gene I "I"P ;I La -1-li li~il,. i 1296 7 0, 0 7 ;00 WO 97/46079 PCT/US97/09682 77 product and, thus, is included within the definition of an antibody. A non-naturally occurring antibody, or fragment thereof, such as a chimeric antibody or humanized antibody also is included within the meaning of the term antibody. Such a non-naturally occurring antibody can be constructed using solid phase peptide synthesis, produced recombinantly or obtained, for example, by screening a combinatorial library consisting of variable heavy chains and variable light chains as described by Huse et al., Science 246:1275-1281 (1989), which is incorporated herein by reference.

An antibody "specific for" a gene product, or that "specifically binds" a gene product, binds with substantially higher affinity to that gene product than to an unrelated gene product. An antibody specific for a gene product also can have specificity for a related gene product. For example, an antibody specific for a Zea mays AP1 gene product also can specifically bind an Arabidopsis thaliana API gene product or a Brassica oleracea AP1 gene product.

An antibody that specifically binds a Zea mays AP1 gene product (SEQ ID NO: for example, can be prepared using a Zea mays AP1 fusion protein or a synthetic peptide encoding a portion of Zea mays AP1 (SEQ ID NO: 8) as an immunogen. One skilled in the art would know that purified Zea mays AP1 gene product, which can be prepared from a natural source or produced recombinantly according to a method of the invention, or a fragment of a Zea mays AP1 gene product, including a peptide portion of Zea mays API such as a synthetic peptide, can be used as an immunogen. For example, preparation of antisera that specifically binds an AP1 gene product is described in Example VI using a GST-AP1 78 fusion protein containing amino acids 190 to 251 of API as an immunogen. In addition, a non-immunogenic fragment ?or synthetic peptide derived from Zea mays AP1, for example, can be made immunogenic by coupling the non-immunogenic fragment or peptide (hapten) to a carrier molecule such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). In addition, various other carrier molecules and methods for coupling a hapten to a carrier molecule are well known in the art as described, for example, by Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1988), which is incorporated herein by reference.

Also provided is an expression vector containing a nucleic acid molecule encoding a floral meristem identity gene product such as API, CAL or LFY operably linked to a heterologous regulatory element.

Expression vectors are well known in the art and provide .a means to transfer and express an exogenous nucleic acid molecule into a host cell. Thus, an expression vector contains, for example, transcription start and stop sites S. such as a TATA sequence and a poly-A signal sequence, as well as a translation start site such as a ribosome binding site and a stop codon, if not present in the coding sequence.

As used herein, the term "heterologous .regulatory element" means a regulatory element derived O"O: from a different gene than the gene encoding the floral meristem identity gene product to which it is operably linked. A vector containing a floral meristem identity gene, however, contains a nucleic acid molecule encoding a floral meristem identity gene product operably linked to a homolgous regulatory element. Such a vector does not contain a nucleic acid molecule encoding a floral i meristem identity gene product operably linked to a 'BR I F; I III f i !Z i, 700 79 heterologous regulatory element and, thus, is not an expression vector of the invention.

SAlso provided is a -plant expression vector containing a floral meristem identity gene product operably linked to a heterologous regulatory element. For example, a plant expression vector containing a nucleic acid molecule encoding an API gene product having at least about 70 percent amino acid identity with an amino acid sequence of Arabidopsis thaliana AP1 (SEQ ID NO: 2) in the region from amino acid 1 to amino acid 163 or with the amino acid sequence of Zea mays AP1 (SEQ ID NO: 8) in the region from amino acid 1 to amino acid 163 is provided. A plant expression vector containing a floral meristem identity gene product operably linked to a constitutive regulatory element, such as the cauliflower mosaic virus 35S promoter, is provided. In addition, a plant expression vector containing a floral meristem identity gene product operably linked to an inducible regulatory element is provided.

A useful plant expression vector can contain a constitutive regulatory element for expression of an exogenous nucleic acid molecule in all or most tissues of a seed plant. The use of a constitutive regulatory element can be particularly advantageous because expression from the element is relatively independent of developmentally regulated or tissue-specific factors.

For example, the cauliflower mosaic virus 35S promoter (CaMV 35S) is a well-characterized constitutive regulatory element that produces a high level of expression in all plant tissues (Odell et al., Nature 313:810-812 (1985), which is incorporated herein by reference). Furthermore, the CaMV 35S promoter can be particularly useful due to its activity in numerous A different seed plant species (Benfey and Chua, Si P. IS Ih, x I-I- i\ 3 11 190 0( WO 97/46079 PCT/US97/09682 250:959-966 (1990), which is incorporated herein by reference; Odell et al., supra, 1985). Other constitutive regulatory elements useful for expression in a seed plant include, for example, the cauliflower mos; virus 19S promoter; the Figwort mosaic virus promoter (Singer et al., Plant Mol. Biol. 14:433 (1990), which is incorporated herein by reference); and the nopaline synthase (nos) gene promoter (An, Plant Physiol. 81:86 (1986), which is incorporated herein by reference).

In addition, an expression vector of the invention can contain a regulated gene regulatory element such as a promoter or enhancer element. A particularly useful regulated promoter is a tissue-specific promoter such as the shoot meristem-specific CDC2 promoter (Hemerly et al., Plant Cell 5:1711-1723 (1993), which is incorporated herein by reference), or the AGL8 promoter, which is active in the apical shoot meristem immediately after the transition to flowering (Mandel and Yanofsky, supra, 1995). The promoter of the SHOOTMERISTEMLESS gene, which is expressed exclusively in the shoot meristem beginning within an embryo and throughout the angiosperm life cycle, also can be a particularly useful tissue-specific gene regulatory element (see Long et al., Nature 379:66-69 (1996), which is incorporated herein by reference).

An appropriate regulatory element such as a promoter is selected depending on the desired pattern or level of expression of a nucleic acid molecule linked thereto. For example, a constitutive promoter, which is active in all tissues, would be appropriate if expression of a gene product in all plant tissues is desired. In addition, a developmentally regulated or tissue-specific regulatory element can be useful to direct floral meristem identity gene expression to specific tissues, WO 97/46079 PCT/US97/09682 81 for example. As discussed above, inducible expression also can be particularly useful to manipulate the timing of gene expression such that, for example, a population of transgenic seed plants of the invention that contain an expression vector comprising a floral meristem identity gene linked to an inducible regulatory element can undergo early reproductive development at essentially the same time. Selecting the time of reproductive development can be useful, for example, in manipulating the time of crop harvest.

Using nucleic acid molecules encoding AP1 provided herein, the skilled artisan can isolate, if desired, a novel ortholog of AP1. For example, one would choose a region of API that is highly conserved among known API sequences such as a region that is highly conserved between Arabidopsis API (SEQ ID NO: 1) and Zea mays API (GenBank accession number L46400; SEQ ID NO: 7) to screen a cDNA or genomic library of interest for a novel AP1 ortholog. One can use a full-length Arabidopsis API (SEQ ID NO: for example, to isolate a novel ortholog of API (see Example If desired, the region encoding the MADS domain, which is common to a number of genes, can be excluded, from the sequence used as a probe. Similarly, the skilled artisan knows that a nucleic acid molecule encoding a full-length CAL cDNA such as Arabidopsis CAL (SEQ ID NO: 9) or Brassica oleracea CAL (SEQ ID NO: 11) can be useful in isolating a novel CAL ortholog.

For example, the Arabidopsis API cDNA (SEQ ID NO: 1) can be used as a probe to identify and isolate a novel API ortholog. Using a nucleotide sequence derived from a conserved region of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, for example, a nucleic acid WO 97/46079 PCT/US97/09682 82 molecule encoding a novel API ortholog can be isolated from other plant species. Using methods such as those described by Purugganan et al., supra, 1995, one can readily confirm that the newly isolated molecule is an API ortholog. Thus, a nucleic acid molecule encoding an AP1 gene product, which has at least about 70 percent amino acid identity with the amino acid sequence of SEQ ID NO: 2 (Arabidopsis AP1) in the region from amino acid 1 to amino acid 163 or with the amino acid sequence of SEQ ID NO: 8 (Zea mays API) in the region from amino acid 1 to amino acid 163 can be isolated and identified using well known methods.

Similarly, in order to isolate an ortholog of CAL, one can choose a region of CAL that is highly conserved among known CAL cDNAs, such as a region conserved between Arabidopsis CAL (SEQ ID NO: 9) and Brassica oleracea CAL (SEQ ID NO: 11). The Arabidopsis CAL cDNA (SEQ ID NO: 9) or Brassica oleracea CAL cDNA (SEQ ID NO: 11), or a nucleotide fragment thereof, can be used to identify and isolate a novel CAL ortholog using methods such as those described in Example V. In order to identify related MADS domain genes, a nucleotide sequence derived from the MADS domain of API or CAL, for example, can be useful to isolate a related gene sequence encoding this DNA-binding motif.

Hybridization conditions for isolating a gene ortholog, for example, are relatively stringent such that non-specific hybridization is minimized. Appropriate hybridization conditions can be determined empirically, or can be estimated based, for example, on the relative G+C content of the probe and the number of mismatches between the probe and target sequence, if known.

Hybridization conditions can be adjusted as desired by 83 varying, for example, the temperature of hybridizing or the salt concentration (Sambrook, supra, 1989) Also provided is a-kit for converting shoot meristem to floral meristem in an angiosperm, which contains a plant expression vector having a nucleic acid molecule encoding a floral meristem identity gene product. A kit for promoting early reproductive development in a seed plant, which contains a plant expression vector having a nucleic acid molecule encoding a floral meristem identity gene product, also is provided. If desired, such kics can contain appropriate reagents to facilitate high efficiency transformation of a seed plant with a plant expression vector of the invention. Furthermore, if desired, a control vector lacking a floral meristem identity gene can be included in the kits to determine, for example, the efficiency of transformation.

The following examples are intended to illustrate but not limit the present invention.

EXAMPLE I o Conversion of shoot meristem to floral meristem and early reproductive development in an APETALA1 transcenic plant This example describes methods for producing a transgenic Arabidopsis plant containing ectopically expressed AP expressed AP1.

\8P. IS1\11-$\ -ha I I\ Sp- i 32 167C1. 1907 0( WO 97/46079 PCT/US97/09682 84 A. Ectopic expression of APETALA1 converts inflorescence shoots into flowers Transgenic plants that constitutively express API from the cauliflower mosaic virus 35S (CaMV promoter were produced to determine whether ectopic API expression was sufficient to convert shoot meristem to floral meristem. The API coding sequence was placed under control of the CaMV 35S promoter (Odell et al., supra, 1985) as follows. Bam HI linkers were ligated to the Hinc II site of the full-length API complementary DNA (Mandel et al., supra, (1992), which is incorporated herein by reference) in pAM116, and the resulting Bam HI fragment was fused to the CaMV 35S promoter (Jack et al., Cell 76:703-716 (1994), which is incorporated herein by reference) in pCGN18 to create pAM563.

Transgenic 35S-API Arabidopsis plants of the Columbia ecotype were generated by selecting kanamycin-resistant plants after Agrobacterium-mediated plant transformation using the in planta method (Bechtold et al., C.R. Acad. Sci. Paris 316:1194-1199 (1993), which is incorporated herein by reference). All analyses were performed in subsequent generations. Approximately 120 independent transgenic lines that displayed the described phenotypes were obtained.

Remarkably, in 35S-AP1 transgenic plants, the normally indeterminate shoot apex prematurely terminated as a floral meristem and formed a terminal flower.

Generally, lateral meristems that normally would produce inflorescence shoots also were converted into solitary flowers. These results demonstrate that ectopic expression of AP1 in shoot meristem is sufficient to convert shoot meristem to floral meristem, even though WO 97/46079 PCT/US97/09682 AP1 normally is not absolutely required to specify floral meristem identity.

B. LEAFY is not required for the conversion of inflorescence shoots to flowers in ah APETALAI transgenic plant To determine whether the 35S-API transgene causes ectopic LFY activity, and whether ectopic LFY activity is required for the conversion of shoot meristem to floral meristem, the 35S-AP1 transgene was introduced into Arabidopsis Ify mutants. The 35S-AP1 transgene was crossed into the strong 1fy-6 mutant background and the F 2 progeny were analyzed.

Mutant Ify plants containing the 35S-AP1 transgene displayed the same conversion of apical and lateral shoot meristem to floral meristem as was observed in transgenics containing wild type LFY. However, the resulting flowers had the typical Ify mutant phenotype, in which floral organs developed as sepaloid and carpelloid structures, with an absence of petals and stamens. These results demonstrate that LFY is not required for the conversion of shoot meristem to floral meristem in a transgenic angiosperm that ectopically expresses AP1.

C. APETALAI is not sufficient to specify organ fate As well as being involved in the early step of specifying floral meristem identity, API also is involved in specifying sepal and petal identity at a later stage in flower development. Although API RNA initially is expressed throughout the young flower primordium, it is WO 97/46079 PCT/US97/09682 86 later excluded from stamen and carpel primordia (Mandel et al., supra, 1992). Since the CaMV 35S promoter is active in all floral organs, 35S-AP1 transgenic plants are likely to ectopically express AP1 in stamens and carpels. However, the normal stamens and carpels in transgenic plants indicate that AP1 is not sufficient to specify sepal and petal organ fate.

D. Ectopic expression of APETALAI causes early reproductive development In addition to its ability to alter inflorescence meristem identity, ectopic expression of API also influences the vegetative phase of plant growth.

Wild-type Arabidopsis plants have a vegetative phase during which a basal rosette of leaves is produced, followed by the transition to reproductive growth. The transition from vegetative to reproductive growth was measured both in terms of the number of days post-germination until the first visible flowers were observed, and by counting the number of leaves. Under continuous light, wild-type and 35S-AP1 transgenic plants flowered after producing 9.88 1.45 and 4.16±0.97 leaves, respectively. Under short-day growth conditions (8 hours light, 16 hours dark, 24 wild-type and transgenic plants flowered after producing 52.42±3.47 and 7.4±1.18 leaves, respectively.

Under continuous light growth conditions, flowers appear on wild-type Arabidopsis plants after approximately 18 days, whereas the 35S-AP1 transgenic plants flowered after an average of only 10 days.

Furthermore, under short-day growth conditions, flowering is delayed in wild-type Arabidopsis plants until approximately 10 weeks after germination, whereas WO 97/46079 PCT/US97/09682 87 transgenic plants flowered in less than about five weeks.

Thus, ectopic API expression significantly reduced the time of reproductive development, as indicated by the time of flowering. Ecotopic API expression also reduced the delay of flowering caused by short day growth conditions.

EXAMPLE II Conversion of inflorescence shoots into flowers in an CAULIFLOWER transgenic plant This example describes methods for producing a transgenic Arabidopsis plant that ectopically expresses

CAL.

Transgenic Arabidopsis plants that ectopically express CAL in shoot meristem were generated. The full-length CAL cDNA was inserted downstream of the CaMV promoter in the Eco RI site of pMON530 (Monsanto Co., St. Louis, Missouri) This plasmid was introduced into Agrobacterium strain ASE and used to transform the Columbia ecotype of Arabidopsis using the modified vacuum infiltration method described by Bechtold et al., supra, 1993. The 96 transgenic lines that harbored the construct had a range of weak to strong phenotypes.

Transgenic plants with the strongest phenotypes (27 lines) had a phenotype that closely resembled the tfl mutant phenotype.

The apical and lateral inflorescence shoots of transgenic plants were converted into flowers.

Furthermore, the 35S-CAL transgenic plants were characterized by early reproductive development, as indicated by an early flowering phenotype. These results WO 97/46079 PCT/US97/09682 88 demonstrate that ectopic expression of CAL is sufficient for the conversion of shoots to flowers and for promoting early reproductive development.

EXAMPLE III Conversion of shoots into flowers and early reproductive developemnt in a LEAFY transgenic plant This example describes methods for producing transgenic Arabidopsis ectopically expressing LFY and transgenic aspen ectopically expressing LFY.

A. Conversion of Arabidopsis shoots and early Arabidopsis reproductive development by LEAFY Transgenic Arabidopsis plants were generated by transforming Arabidopsis with LFY under the control of the CaMV 35S promoter (Odell et al., supra, (1985)). A LFY complementary DNA (Weigel et al, Cell 69:843-859 (1992), which is incorporated herein by reference) was inserted into a T-DNA transformation vector containing a CaMV 35S promoter and a 3' nos cassette (Jack et al., supra, 1994). Transformed seedlings were selected for kanamycin resistance. Several hundred Arabidopsis transformants in three different genetic backgrounds (Nossen, Wassilewskija and Columbia) were recovered, and several lines were characterized in detail.

High levels of LFY RNA expression were detected by northern blot analysis in 35S-LFY transgenics. In general, Nossen lines had weaker phenotypes, especially when grown under short day conditions. The transgene of line DW151.117 (ecotype Wassilewskija) was WO 97/46079 PCTIUS97/09682 89 introgressed into the erecta background by backcrossing to a Landsberg erecta strain. Plants were grown under 16 hours light and 8 hours dark. The 35S-LFY transgene provided at least as much LFY activity as the endogenous gene and completely suppressed the Ify mutant phenotype when crossed into the background of the Ify-6 null allele.

Most 35S-LFY transgenic plants lines demonstrated a very similar, dominant and heritable phenotype. Secondary shoots that arose in lateral positions were consistently replaced by solitary flowers, and higher-order shoots were absent. Although the number of rosette leaves was unchanged from the wild type, plants flowered earlier than wild type: the solitary flowers in the axils of the rosette leaves developed and opened precociously. In addition, the primary shoot terminated with a flower. In transgenics having the most extreme phenotypes, a terminal flower was formed immediately above the rosette.

This gain of function phenotype (conversion of shoots to flowers) is the opposite of the Ify loss of function phenotype (conversion of flowers to shoots). These results demonstrate that LFY encodes a developmental switch that is both sufficient and necessary to convert shoot meristem to flower meristem in an angiosperm.

The effects of constitutive LFY expression differ for primary and secondary shoot meristems.

Secondary meristems were transformed into flower meristem, apparently as soon as it developed, and produced only a single, solitary flower. In contrast, primary shoot meristem produced leaves and lateral flowers before being consumed in the formation of a terminal flower. These developmental differences WO 97/46079 PCT/US97/09682 indicate that a meristem must acquire competence to respond to the activity of a floral meristem identity gene such as LFY.

B. Conversion of aspen shoots by LEAFY Given that constitutive expression of LFY induced early reproductive development as indicated by precocious flowering during the vegetative phase of Arabidopsis, the effect of LFY on the flowering of other seed plant species was examined. The perennial tree, hybrid aspen, is derived from parental species that flower naturally only after 8-20 years of growth (Schopmeyer USDA Agriculture Handbook 450: Seeds of Woody Plants in the United States, Washington DC, USA: US Government Printing Office. pp. 645-655 (1974)).

35S-LFY transgenic aspen plants were obtained by Agrobacterium-mediated transformation of stem segments and subsequent regeneration of transgenic shoots in tissue culture.

Hybrid aspen was transformed exactly as described by Nilsson et al. (Transgen. Res. 1:209-220 (1992), which is incorporated herein by reference).

Levels of LFY RNA expression were similar to those of Arabidopsis, as determined by northern blot analysis. The number of vegetative leaves varied between different regenerating shoots, and those with a higher number of vegetative leaves formed roots, allowing for transfer to the greenhouse. Individual flowers were removed either from primary transformants that had been transferred to the greenhouse, or from catkins collected in spring, 1995, at Carlshem, Umed, Sweden) from a tree whose age was determined by counting the number of annual rings in a core extracted with an increment borer at meters above ground level. Flowers were fixed in WO 97/46079 PCT/US97/09682 91 formaldehyde/acetic acid/ethanol and destained in ethanol before photography.

The overall phenotype of 35S-LFY transgenic aspen was similar to that of 35S-LFY Arabidopsis. In wild-type plants of both species, flowers normally are formed in lateral positions on inflorescence shoots. In aspen, these inflorescence shoots, called catkins, arise from the leaf axils of adult trees. In both Arabidopsis and 35S-LFY aspen, solitary flowers were formed instead of shoots in the axils of vegetative leaves. Moreover, as in Arabidopsis, the secondary shoots of transgenic aspen were more severely affected than the primary shoot.

Regenerating 35S-LFY aspen shoots initially produced solitary flowers in the axils of normal leaves.

However, the number of vegetative leaves was limited, and the shoot meristem was prematurely consumed in the formation of an aberrant terminal flower. Early reproductive development as demonstrated by precocious flowering was specific to 35S-LFY transformants and was not observed in non-transgenic controls. Furthermore, not a single instance of precocious flower development has been observed in more than 1,500 other lines of transgenic aspen generated with various constructs from 1989 to 1995 at the Swedish University of Agricultural Sciences. These results demonstrate that a floral meristem identity gene product can promote early reproductive development in a heterologous angiosperm species.

WO 97/46079 PCT/US97/09682 92 EXAMPLE IV Dexamethasone-inducible floral meristem identity gene activity in transgenic plants This example describes the construction and characterization of an APl-glucocorticoid receptor ligand binding domain chimera and its dexamethasone-inducible activity in Arabidopsis.

A. Construction and characterization of an API-glucocorticoid receptor ligand binding domain chimera A nucleic acid molecule encoding an AP1-glucocorticoid receptor ligand binding domain chimera was prepared as follows. Primers corresponding to the translation initiation and termination codons of AP1 were synthesized for PCR amplification of the Arabidopsis API cDNA. Primer 5'-GGATCCGGATCAAAAAIGGGAAGGGGTAG-3' (SEQ ID NO: 25) contains a translation initiation codon, which is indicated by underlining. Primer 5'-GGATCCGCTGCGGCGAAGCAGCCAAGGTTG-3' (SEQ ID NO: 26) contains a modified translation termination site, which is indicated by underlining and allows the nucleic acid molecule encoding API to be linked in frame to the nucleic acid molecule encoding the glucocorticoid receptor (GR) ligand binding domain.

The full length Arabidopsis AP1 cDNA in pAM116 (see Example I) was used as the template for PCR amplification with primers SEQ ID NOS: 25 and 26, each of which contain a Bam H1 site. The resulting Bam HI fragment, which encodes the full-length Arabidopsis AP1 cDNA except for the translation termination codon, was cloned into the unique Bar HI site of the GR fusion vector constructed by Lloyd et al., supra, 1994. DNA WO 97/46079 PCT/US97/09682 93 sequence analyses confirmed that the construct contained the predicted nucleotide sequence.

The resulting AP1-GR construct was introduced into Agrobacterium strain ASE, and apl-15 mutant plants were transformed using the vacuum infiltration method described in Example I. Approximately 100 independently derived lines were selected in kanamycin for further analysis.

B. Dexamethasone-inducible activity of an AP1-glucocorticoid receptor ligand binding domain chimera in Arabidopsis Kanamycin-resistant transgenic Arabidopsis lines are analyzed in subsequent generations for AP1 activity. After application of dexamethasone to transgenic plants, AP1 activity is monitored by visual inspection for 1) flowering that is earlier than wild-type or 2) partial or complete rescue of the apl mutant phenotype.

To assay for dexamethasone-inducible activity, plants are watered with varying concentrations of dexamethasone. A range of dexamethasone concentrations are tested to determine overall levels of API activity and to determine the resulting phenotypes. A concentration of 1 gM or less dexamethasone preferably is used for induction of AP1 activity.

In addition, dexamethasone is applied directly to plants by spraying. Spraying, like watering, leads to a significant induction of API activity, resulting in the corresponding rescue of the apl mutant phenotype and early reproductive development. Although a single application of dexamethasone is sufficient to increase WO 97/46079 PCT/US97/09682 94 API activity and promote early reproductive development, dexamethasone is applied either once, or repeatedly, and the treatments compared for any observed differences under long or short day conditions as disclosed below.

Dexamethasone is applied to plants at various times post-germination. For example, a large number of AP1-GR transgenic Arabidopsis plants are grown, some of which are treated with dexamethasone on day 1 post-germination, some on day 2, etc., all the way up until and beyond the time at which Arabidopsis plants normally flower. These analyses include plants grown under long day, short day, and under a variety of temperatures. For example, Arabidopsis plants, which typically are grown at 25 0 C, also can be analyzed for AP1 activity at 20 0 C and 15 0 C (see, for example, Bowman et al.

Arabidopsis: An Atlas of Morphology and Development, New York: Springer (1994), which is incorporated by reference herein).

EXAMPLE V Identification and characterization of the Zea mays APETALAI cDNA This example describes the isolation and characterization of Zea mays ZAP1 complementary DNA, which is an ortholog of the Arabidopsis floral meristem identity gene API.

A. Identification and characterization of a nucleic acid sequence encoding ZAP1 The utility of using a cloned floral homeotic gene from Arabidopsis to identify the putative ortholog in maize has been demonstrated previously (Schmidt et WO 97/46079 PCT/US97/09682 al., supra, (1993), which is incorporated herein by reference). As described in Mena et al. (Plant J.

8(6):845-854 (1995)), the maize ortholog of the Arabidopsis API floral meristem identity gene, was isolated by screening a Zea mays ear cDNA library using the Arabidopsis API cDNA (SEQ ID NO: 1) as a probe. A cDNA library was prepared from wild-type immature ears as described by Schmidt et al., supra, 1993, and screened using the Arabidopsis API cDNA SEQ ID NO: 1 as the probe.

Low-stringency hybridizations with the API probe were conducted as described previously for the isolation of ZAGI using the AG cDNA as a probe (Schmidt et al., supra, 1993). Positive plaques were isolated and cDNAs were recovered in Bluescript by in vivo excision.

Double-stranded sequencing was performed using the Sequenase Version 2.0 kit Biochemical, Cleveland, Ohio) according to the manufacturer's protocol.

The nucleotide sequence and deduced amino acid sequence of the ZAPI cDNA are provided as SEQ ID NOS: 7 and 8. The deduced amino acid sequence for ZAPI shares 89% identity with Arabidopsis API through the MADS domain (amino acids 1 to 57) and 70% identity through the first 160 amino acids, which includes the K domain. The high level of amino acid sequence identity between ZAPI and API (SEQ ID NOS: 8 and as well as the expression pattern of ZAPI in maize florets (see below), indicate that ZAPI is the maize ortholog of Arabidopsis API.

B. RNA expression pattern of ZAPI Total RNA was isolated from different maize tissues as described by Cone et al., Proc. Natl, Acad.

Sci.. USA 83:9631-9635 (1986), which is incorporated WO 97/46079 PCTIUS97/09682 96 herein by reference. RNA was prepared from ears or tassels at early developing stages (approximately 2 cm in size), husk leaves from developing ear shoots, shoots and roots of germinated seedlings, leaves from 2 to 3 week old plants and endosperm, and embryos at 18 days after pollination. Mature floral organs were dissected from ears at the time of silk emergence or from tassels at several days pre-emergence. To study expression patterns in the mature female flower, carpels were isolated and the remaining sterile organs were pooled and analyzed together. In the same way, stamens were dissected and collected from male florets and the remaining organs (excluding the glumes) were pooled as one sample.

RNA concentration and purity was determined by absorbance at 260/280 nM, and equal amounts (10 Mg) were fractionated on formaldehyde-agarose gels. Gels were stained in a solution of 0.125 Mg ml 1 acridine orange to confirm the integrity of the RNA samples and the uniformity of gel loading, then RNA was blotted on to Hybond-N® membranes (Amersham International, Arlington Heights, Illinois) according to the manufacturer's instructions. Prehybridization and hybridization solutions were prepared as previously described (Schmidt et al., Science 238:960-963 (1987), which is incorporated herein by reference). The probe for ZAPI RNA expression studies was a 445 bp Sac I/ Nsi I fragment from the 3' end of the cDNA. Southern blot analyses were conducted to establish conditions for specific hybridization of this probe. No cross-hybridization was detected using hybridization at 60 0 C in 50% formamide and washes at in 0.1x SSC and 0.5% SDS.

The strong sequence similarity between ZAP1 and API indicated that ZAPI was the ortholog of this Arabidopsis floral meristem identity gene. As a first WO 97/46079 PCT/US97/09682 97 approximation of whether the pattern of ZAPI expression paralleled that of API, a blot of total RNA from vegetative and reproductive organs was hybridized with a gene-specific fragment of the ZAPI cDNA (nucleotides 370 to 820 of SEQ ID NO: ZAP1 RNA was detected only in male and female inflorescences and in the husk leaves that surround the developing ear. No ZAP1 RNA expression was detectable in RNA isolated from root, shoot, leaf, endosperm, or embryo tissue. The restriction of ZAP1 expression to terminal and axillary inflorescences is consistent with ZAP1 being the Arabidopsis API ortholog.

Male and female florets were isolated from mature inflorescences, and the reproductive organs were separated from the remainder of the floret. RNA was isolated from the reproductive and the sterile portions of the florets. ZAP1 RNA expression was not detected in maize stamens or carpels, whereas high levels of ZAPI RNA were present in developing ear and tassel florets from which stamens and carpels had been removed. Thus, the exclusion of ZAP1 expression from stamens and carpels and its inclusion in the RNA of the non-reproductive portions of the floret (lodicules, lemma and palea) is similar to the pattern of expression of API in flowers of Arabidopsis.

EXAMPLE VI Production and characterization of anti-AP1 antisera This example demonstrates the production and characterization of antisera that specifically binds the Arabidopsis AP1 gene product.

WO 97/46079 PCT/US97/09682 98 Western blotting was performed with plant tissue extracts and crude antisera from rabbits immunized with a GST-AP1 fusion protein encoding amino acids 190 to 251 of Arabidopsis thaliana API (SEQ ID NO: The C-terminal region of AP1 spanning amino acids 190 to 251 does not include the MADS domain, which is common to a number of proteins. As shown in Figure 1, the anti-API sera reacted with a 90 kDa protein in inflorescence tissue extracts prepared from wild type Arabidopsis thaliana (Landsburg ecotype). As expected, this reactivity was absent from Arabidopsis mutants lacking API such as apl-1 or apl-15 (compare lanes 3 and 4 to lane 2).

AP1 expression was reduced significantly in inflorescence tissue extracts from the Arabidopsis ap2-2 mutant as compared to wild type plants, indicating that AP2 normally functions to increase or maintain the level of API RNA or protein expression (see lanes 2 and Similarly, reduced AP1 expression in ify-6 mutant plants indicates that LFY normally functions to enhance expression of AP1 at the RNA or protein level (see lanes 2 and In contrast to the results seen in ap2-2 or 1fy-6 mutant inflorescences, AP1 protein expression in inflorescence tissue from ag-2 mutants is enhanced strikingly as compared to the level of AP1 protein seen in wild type inflorescences (see lanes 1 and These results indicate that the AGAMOUS gene product (AG) negatively regulates AP1 RNA or protein expression.

Western analysis further demonstrated that AP1 protein expression is specific to inflorescence tissue since API reactivity is absent from leaf tissue prepared from wild type Arabidopsis plants (Columbia ecotype; lane In transgenic plants constitutively expressing WO 97/46079 PCT/US97/09682 99 API from the CaMV promoter, however, API protein expression readily was detectable in leaf tissue as shown in lane 8. Reactivity of the anti-AP1 antisera in transgenic leaves but not in wild type Arabidopsis leaves confirmed the specificity of the anti-AP1 sera.

Specificity of the anti-API sera also was demonstrated by specific binding of the antisera to API but not to the closely related CAL gene product. For example, inflorescence tissue extract from an apl-1 or mutant plant (lane 3 or 4, respectively), which contains CAL but not API, was not reactive with the anti-AP1 rabbit sera. These data indicate that the anti-AP1 sera does not react with the CAL gene product.

For production of anti-AP1 sera, a Sty I fragment of the Arabidopsis thaliana API cDNA, which encodes amino acids 190 to 251, was gel purified, blunt ended with Klenow fragment and ligated into the Sma I site of pGEX3X (Pharmacia, Piscataway, NJ) to make pGEX-AP1 190 251 for expression of a GST-AP1 190 25 fusion protein. DH5a E. coli were transformed with the resulting vector by standard techniques (Sambrook, supra, 1989).

A bacterial culture of a pGEX-AP1 190 -2 51 transformant was grown to an OD 600 of 0.5, and GST-AP1 190 25 1 expression was induced by addition of 1mM IPTG. The GST-AP1 190 251 bacterial pellet was harvested after three hours growth at 37 0 C, washed once with phosphate-buffered saline (PBS; pH 7.2) and lysed by two cycles of freeze-thawing. The cell lysate was resuspended in one-fiftieth of the culture volume in ice cold EB (2 mM EDTA, 2mM DTT, 1 mM PMSF, 5 gg/ml leupeptin, 7.5 yg/ml pepstatin, 1% aprotinin in PBS pH 7.2) with 2 mg/ml WO 97/46079 PCT/US97/09682 100 lysozyme and incubated on ice for 30 minutes. Triton X-100 was added to and the solution was sonicated mildly. The extract was clarified by two successive centrifugations of 1 and 15 minutes, respectively, at 13,000 x g in a microfuge.

The GST-AP1 19 0- 251 fusion protein was purified from the bacterial extract as follows.

Glutathione-Sepharose beads (150 which had been pre-equilibrated in EB with 1% Triton X-100, were added to 1 ml of soluble extract in an Eppendorf tube and incubated on a rotating wheel for 60 minutes at 4 0 C. The beads were washed five times in 1 ml EB with 1% Triton X-100; resuspended in protein sample buffer and loaded on a preparative SDS-PAGE gel (Laemmli, Nature 227:680-685 (1970), which is incorporated herein by reference).

Following electrophoresis, the gel was stained for five minutes in 0.05% Coomassie R250 (Fisher Scientific, Pittsburgh, Pennsylvania) in distilled water and subsequently destained in distilled water. GST-AP1 1 g 90 251 fusion protein was cut out of the gel and electroeluted in 0.5X transfer buffer for 3 hours at 100V as described in Harlow and Lane, supra, 1988. The GST-AP 1 90 251 fusion protein was emulsified with Freund's adjuvant and injected into rabbits by Immunodynamics (La Jolla, CA) Crude rabbit serum was used for western analysis at a dilution of 1 to 2000. Binding was detected using a secondary antibody coupled to peroxidase (Promega, Madison, WI; 1 to 2500 dilution) and revealed using an enhanced chemiluminesence kit (Amersham).

Plant protein extracts for western analysis were prepared by homogenizing 100 gl plant tissue with 200 Ip 2XFSB (Laemmli, supra, 1970) in a Kontes microfuge tube with a pistil. The extract was denatured in boiling water bath for 5 minutes, sonicated for 1 minute and WO 97/46079 PCT/US97/09682 101 clarified by two successive spins of 5 and 15 minutes in a microfuge at 13'000 x g prior to electophoresis.

EXAMPLE VII Cosuppression of API activity This example demonstrates the use of cosuppression to inhibit endogenous AP1 activity in Arabidopsis.

The full length API cDNA from pAM116 (see Example I) was inserted into the Eco RI site of pMON530, and the resulting construct was introduced into Agrobacterium strain ASE. Wild type Arabidopsis was transformed as described in Example I and analyzed for apl mutant phenotypes. In this way, a large number of independently generated cosuppressed lines were generated. Each of the cosuppressed lines had a phenotype similar or identical to apl-1 mutant plants, which lack AP1 activity, indicating that the activity of both the introduced and endogenous copies of API was suppressed. Analysis of API expression levels by RNA in situ hybridization demonstrated that API expression was reduced and delayed in the cosuppressed transgenic lines having the apl mutant phenotype. Futhermore, in a samll fraction of the cosuppressed transgenic lines, a enhanced phenotype resembling the cauliflower phenotype was observed. This enhanced phenotype indicated that introduction of an API construct can supress expression of both endogenous API and CAL.

WO 97/46079 PCT/US97/09682 102 Although the invention has been described with reference to the examples above, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

WO 97/46079 PCT/US97/09682 103 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: The Regents of the University of California (ii) TITLE OF INVENTION: Seed Plants Exhibiting Inducible Early Reproductive Development and Methods of Making Same (iii) NUMBER OF SEQUENCES: 26 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Campbell Flores LLP STREET: 4370 La Jolla Village Drive, suite 700 CITY: San Diego STATE: California COUNTRY: USA ZIP: 92122 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: Campbell, Cathryn A.

REGISTRATION NUMBER: 31,815 REFERENCE/DOCKET NUMBER: FP-UD 2629 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (619) 535-9001 TELEFAX: (619) 535-8949 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 1057 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATION: 124..893 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1..1057 OTHER INFORMATION: /note= "product Arabidopsis thaliana AP1." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: CTTTCCAATT GGTTCATACC AAAGTCTGAG CTCTTCTTTA TATCTCTCTT GTAGTTTCTT ATTGGGGGTC TTTGTTTTGT TTGGTTCTTT TAGAGTAAGA AGTTTCTTAA AAAAGGATCA WO 97/46079 WO 9746079PCTfUS97/09682 104 AGG AAA~

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Ser

AAG

Lys

GAG

GiU

TCT

Ser

CAG

Gin 185

CCG

Pro

TCT

Ser

GC.A

Ala

TGC

Arg Ile GCT GGT Ala Gly GAA GTT Giu Val TCC ACT Ser Thr TCT TAC Ser Tyr AAC TGG Asn Trp 90 GAG AGA Giu Arg CCT AAA Pro Lys CAC ATC His Ile CTC CAA Leu Gin 155 AAA CAG Lys Gin 170 TGG GAT Tr Asp CAG CAG Gin Gin CCT TTT Pro Phe *ATG AGG *Met Arg 235 AAC CTT

CTT

Leu

GCT

GAT

Asp

GCC

Al a

TCG

Ser

A.AC

Asn

GAG

Giu

CGC

Arg 140

AAA

Lys

ATC

Ile

CAG

Gin

CAC

His

CTC

Leu 220

AGG

Arg

GGC

TTG.

Leu

CTT

Leu

TCT

Ser

GAA.

Giu

ATG

Met

CAG

Gin

CTT

Leu 125

ACT

Thr

AAG

Lys

AAG

Lys

CAG

Gin

CAA

Gin 205

AAC

Asn

A&T

Asn

TGC

PAAG

Liys

GTT

Val

TGT

Cys

AGA

Arg

GAG

Giu

AGG

Arg 110

CAG

Gin

AGA

Arg

GAG

Giu

GAG

Glu

AAC

As n 190

ATC

Ile

ATG

met

GAT

ASP

TTC

AAA

Lys

GTC

Vai

ATG

met

CAG

Gin

TAT

Tyr

CAT

His

AAT

As n

AAA

LySB

AAG

Lys

AGG

Arg 175

CAA

Gin

CAG

Gin

GGT

Gly

CTC

Leu

GCC

216 264 312 360 408 456 504 552 600 648 696 744 792 840 888 Leu Thr Leu Giu Pro Val Tyr Asn Cys Asfl Leu Gly CyS Phe Aia GCA TG AAGCATTTCC ATATATATAT TTGTAATCGT Ala CAACA-ATAAA AACAGTTTGC 943

I,

WO 97/46079 WO 9746079PCTfJS97/09682 105 CACATACATA TAAATAGTGG CTAGGCTCTT TTCATCCAAT TAATATATTT TGGCAAATGT TCGATGTTCT TATATCATCA TATATAAATT AGCAGGCTCC TTTCTTTTTT TGTA INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 256 amino acids TYPE: amino acid TOPOLOGY: linear 1003 1057 (ii) MOLECULE TYPE: protein (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Gly Arg Gly Arg Gin His GlU Ser His Lys Ile Ile Ala Arg Leu Leu Gly Glu Gin 130 Gin Leu 145 Ile Gin Lys Ile His Asn Pro Tyr 210 Leu Tyr 225 Val Thr Ile Ser Lys Gly Leu Giu Pro Giu Lys Ala 100 Glu Asp 115 Gin Leu Met Tyr GiU Gin Leu Arg 180 Met Pro 195 Met Leu Gin GIU Arg Phe Vai Lys Arg Ser Lys Leu Asp Giu As n 165 Ala Pro Ser Asp Val Gin Leu Lys Ser Leu Leu Tyr 70 Asp Ile Gin Thr Ser 150 Ser Gin Pro His Asp 230 Lys Cys Phe 55 Giu Val Giu Ala Ala 135 Ile Met Gin Leu Gin 215 Pro Arg Asp 40 Giu.

Arg As n Lau Met 120 Leu Asn Leu Giu Pro 200 Pro Met Arg 25 Aia Tyr Tyr Thr Leu 105 Ser Lys Giu Ser Gin 185 Pro Ser Ala Arg Ile Giu 10 Aia Gly Leu GiU Val Aia Ser Thr Asp Ser Tyr Aia 75 Asn Trp Ser 90 GiU Arg Asn Pro Lys GiU His Ile Arg 140 Leu Gin Lye 155 Lys Gin Ile 170 Trp Asp Gin Gin Gin His Pro Phe Leu 220 Met Arg Arg 235 Asn Lau Leu Ser Glu Met Gin Leu 125 Thr Lys Lys Gin Gin 205 Asn Asn Lys Lys Vai Cys Arg GiU Arg 110 Gin Arg GiU Giu Asn 190 Ile Met Asp Ile Lys Vai Met Gin Tyr His As 0 Lys Lys Arg 175 Gin Gin Gly Lau As n Ala Phe Giu Leu As n Tyr Leu As n Ala 160 Glu Gly His Gly Giu 240 Leu Thr Leu Giu Pro 245 Val Tyr Asn Cys Asn 250 Leu Gly Cys Phe Ala Ala 255 WO 97/46079 WO 9746079PCTIUS97/09682 106 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 794 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 36. .794 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1. .79 4 OTHER INFORMATION: /note=

API...

"Product Brassica oleracea (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TCTTAGAGGA AATAGTTCCT TTAAAAGGGA TAAAA ATG GGA AGG GGT AGG GTT Met Gly Arg Gly Arg Val 1 CAG TTG AAG Gin Leu Lye AAA AGA AGA Lye Arg Arg

AGG

Arg ATA GAA AAC AAG Ile GiU Asn Lys AAT AGA CAA~ GTG Asn Arg Gin Val ACA TTC TCG Thr Phe Her GCT GOT CTT ATG Ala Gly Leu Met AAG AAA GCT CAT GAG ATC TCT GTT CTG Lye Lye Ala His Glu Ile Her Val Leu 30 TOT OAT CYS ASP GCT GAAi OTT GCG Ala Giu Val Ala

CTT

Leu GTT GTC TTC TCC Val Val Phe Her AAG GGG AAA CTC Lys Gly Lys Leu

TTT

Phe GAA TAC TCC ACT Oiu Tyr ser Thr TCT TOT ATO GAG Ser Cys met GlU

AAG,

Lye ATA CTT GAA CGC Ile Lau Glu Arg GAG AGA TAC TCT Giu Arg Tyr Ser

TAC

Tyr GCC GAG AGA CAG Ala Glu Arg Gin ATA GCA CCT GAG Ile Ala Pro Glu TCC GAC Her Asp TCC AAT ACG Her Asn Thr GAG CTT TTG Giu Leu Leu 105

AAC

Asn TGG TCG ATG GAG Trp Ser Met Glu

TAT

Tyr 95 AAT AGG CTT AAG Asn Arg Len Lys GCT AAG ATT Ala Lye Ile 100 GAC TTG CAA Asp Leu Gin 341 389 GAG AGA AAC CAG Gin Arg Aen Gin

AGG

Arg 110 CAC TAT CTT GGG His Tyr Len Gly OCA ATO Ala Met 120 AGC CCT AAG GAA Ser Pro LYe Glu CAG AAT CTA GAG Gin Asn Len Gin CAG CTT OAT ACT Gin Leu ASP Thr

OCT

Ala 135 CTT AAG CAC ATC Len Lys His Ile TCT AGA AAA AAC Her Arg Lye Asn

CAA

Gin 145 CTT ATG TAC GAC Leu Met Tyr Asp

TCC

Her 150 ATC A.AT GAG CTC Ile Asn Glu Len

CAA

Gin 155 AGA AAG GAG AAA Arg Lye Gin Lye ATA CAG GAA CAA Ile Gin Gin Gin AAC AOC Aen Her 165 WO 97/46079 PCT/US97/09682 107 ATG CTT TCC AAG CAG ATT AAG GAG AGG GAA AAC GTT CTT AGG GCG CAA 581 Met Leu Ser Lys Gin Ile Lys Glu Arg Glu Asn Val Leu Arg Ala Gin 170 175 180 CAA GAG CAA TGG GAC GAG CAG AAC CAT GGC CAT AAT ATG CCT CCG CCT 629 Gin Glu Gin Trp Asp Glu Gin Asn His Gly His Asn Met Pro Pro Pro 185 190 195 CCA CCC CCG CAG CAG CAT CAA ATC CAG CAT CCT TAC ATG CTC TCT CAT 677 Pro Pro Pro Gin Gin His Gin Ile Gin His Pro Tyr Met Leu Ser His 200 205 210 CAG CCA TCT CCT TTT CTC AAC ATG GGG GGG CTG TAT CAA GAA GAA GAT 725 Gin Pro Ser Pro Phe Leu Asn Met Gly Gly Leu Tyr Gin Glu Glu Asp 215 220 225 230 CAA ATG GCA ATG AGG AGG AAC GAT CTC GAT CTG TCT CTT GAA CCC GGT 773 Gln Met Ala Met Arg Arg Asn Asp Leu Asp Leu ser Leu Glu Pro Gly 235 240 245 TAT AAC TGC AAT CTC GGC TGC 794 Tyr Asn Cys Asn Leu Gly Cys 250 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 253 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Gly Arg Gly Arg Val Gin Leu Lys Arg Ile Glu Asn Lys Ile Asn 1 5 10 Arg Gln Val Thr Phe Ser Lys Arg Arg Ala Gly Leu Met Lys Lys Ala 25 His Glu Ile Ser Val Leu Cys Asp Ala Glu Val Ala Leu Val Val Phe 40 Ser His Lys Gly Lys Leu Phe Glu Tyr Ser Thr Asp Ser Cys Met Glu 55 Lys Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu Arg Gin Leu 70 75 Ile Ala Pro Glu Ser Asp Ser Asn Thr Asn Trp Ser Met Glu Tyr Asn 90 Arg Leu Lys Ala Lys Ile Glu Leu Leu Glu Arg Asn Gin Arg His Tyr 100 105 110 Leu Gly Glu Asp Leu Gin Ala Met Ser Pro Lys Glu Leu Gin Asn Leu 115 120 125 Glu Gin Gin Leu Asp Thr Ala Leu Lys His Ile Arg Ser Arg Lys Asn 130 135 140 Gin Leu Met Tyr Asp Ser Ile Asn Glu Leu Gin Arg Lys Glu Lys Ala 145 150 155 160 4. I, WO 97/46079 PCT/US97/09682 108 Ile Gin Glu Gin Asn Ser Met Leu Ser Lys Gin Ile Lys Glu Arg Glu 165 170 175 Asn Val Leu Arg Ala Gin Gin Glu Gin Trp Asp Glu Gin Asn His Gly 180 185 190 His Asn Met Pro Pro Pro Pro Pro Pro Gin Gin His Gin Ile Gin His 195 200 205 Pro Tyr Met Leu Ser His Gin Pro Ser Pro Phe Leu Asn Met Gly Gly 210 215 220 Leu Tyr Gin Glu Glu Asp Gin Met Ala Met Arg Arg Asn Asp Leu Asp 225 230 235 240 Leu Ser Leu Glu Pro Gly Tyr Asn Cys Asn Leu Gly Cys 245 250 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 768 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..766 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1..768 OTHER INFORMATION: /note= "product Brassica oleracea var. botrytis API." (xi) SEQUENCE DESCRIPTION: SEQ ID ATG GGA AGG GGT AGG GTT CAG TTG AAG AGG ATA GAA AAC AAG ATC AAT Met Gly Arg Gly Arg Val Gin Leu Lys Arg Ile Glu Asn Lys Ile Asn 1 5 10 AGA CAA GTG ACA TTC TCG AAA AGA AGA GCT GGT CTT ATG AAG AAA GCT Arg Gin Val Thr Phe Ser Lys Arg Arg Ala Gly Leu Met Lys Lys Ala 25 CAT GAG ATC TCT GTT CTG TGT GAT GCT GAA GTT GCG CTT GTT GTC TTC His Glu Ile Ser Val Leu Cys Asp Ala Glu Val Ala Leu Val Val Phe 40 TCC CAT AAG GGG AAA CTC TTT GAA TAC CCC ACT GAT TCT TGT ATG GAG Ser His Lys Gly Lys Leu Phe Glu Tyr Pro Thr Asp Ser Cys Met Glu 55 GAG ATA CTT GAA CGC TAT GAG AGA TAC TCT TAC GCC GAG AGA CAG CTT Glu Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu Arg Gin Leu 70 75 ATA GCA CCT GAG TCC GAC TCC AAT ACG AAC TGG TCG ATG GAG TAT AAT Ile Ala Pro Glu Ser Asp Ser Asn Thr Asn Trp Ser Met Glu Tyr Asn 90 WO 97/46079 PCT/US97/09682 AGG CTT Arg Leu CTT GGG Leu Gly GAG CAA G1u Gin 130 CAA CTT Gin Leu 145 ATA CAG Ile Gin AAC GTT Asn Val CAT AAT His Asn CCT TAC Pro Tyr 210 CTG TAT Leu Tyr 225 CTG TCT Leu Ser

GA

AAG

Lys

GAA

Giu 115

CAG

Gin

ATG

Met

GAA

GlU

CTT

Leu

ATG

Met 195

ATG

Met

CAA

Gin

CTT

Leu

GCT

Ala 100

GAC

Asp

CTT

Leu

TAC

Tyr

CAA

Gin

AGG

Arg 180

CCT

Pro

CTC

Leu

GAA

Giu

GAA

Giu AAG ATT Lys Ile TTG CAA Leu Gin GAT ACT Asp Thr GAC TCC Asp ser 150 AAC AGC Asn ser 165 GCG CAA Ala Gin CCG CCT Pro Pro TCT CAT Ser His GAA GAT GiU Asp 230 CCC GTT Pro Vai 245

GAG

Giu

GCA

Ala

GCT

Ala 135

ATC

Ile

ATG

Met

CAA

Gin

CCA

Pro

CAG

Gin 215

CAA

Gin

TAC

Tyr CTT TTG GAG Leu LeU GiU 105 ATG AGC CCT Met Ser Pro 120 CTT AAG CAC Leu Lys His AAT GAG CTC Asn GiU Leu CTT TCC AAG Leu Ser Lys 170 GAG CAA TGG Giu Gin Trp 185 CCC CCG CAG Pro Pro Gin 200 CCA TCT CCT Pro Ser Pro ATG GCA ATG met Ala Met 109

AGA

Arg

AAG

LyS

ATC

le

CMA

Gin 155

CAG

Gin

GAC

Asp

CAG

Gin

TTT

Phe

AGG

Arg 235

AAC

Asn

GAA

GlU

CGC

Arg 140

AGA

Arg

ATT

Ile

GAG

Giu

CAT

His

CTC

Leu 220

AGG

Arg

AGG

Arg 110

CAG

Gin

AGA

Arg

GAG

GiU

GAG

GiU

AAC

Asn 190

ATC

Ile

ATG

met

GAT

Asp

CAC

His

AAT

Asn

AAA

Lys

AAA

Lys

AGG

Arg 175

CAT

His

CAG

Gin

GGA

Giy

CTC

Leu

TAT

Tyr

CTA

Leu

AAC

Asn

GCC

Ala 160

GMA

Glu

GGC

Gly

CAT

His

GGG

Gly

GAT

Asp 240 336 384 432 480 528 MAC TGC AAC CTT GGC CGT CGC TGC T Asn Cys Asn Leu Gly Arg Arg Cys 250 255 INFORMATION FOR SEQ ID, NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 255 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Gly Arg Giy Arg Val Gin Leu Lys Arg Ile Giu Asn Lys Ile Asn 1 5 10 Arg Gin Val Thr Phe Ser Lys Arg Arg Ala Gly Leu Met Lys Lys Ala 25 His Giu Ile Ser Val Leu Cys Asp Ala Giu Val Ala Leu Val Val Phe 40 Ser His Lys Giy Lys Leu Phe Giu Tyr Pro Thr Asp Ser Cys Met Glu 55 WO 97/46079 WO 9746079PCTIUS97/09682 Giu Ile Arg LeU Glu Gin 145 Ile Asn His Pro Leu 225 Leu Ile Leu Glu Arg Aia Leu Giy Gin 130 Leu Gin Val Asn Tyr 210 Tyr Ser Pro Lys Giu 115 Gin Met Giu Leu Met 195 met Gin Leu GlU Ala 100 Asp Leu Tyr Gin Arg 180 Pro Leu Glu Giu Ser Lys Leu Asp Asp As n 165 Ala Pro Ser Giu Pro 245 Tyr 70 Asp Ile Gin Thr Ser 150 Ser Gin Pro His Asp 230 Giu Arg Tyr Ser Ser Giu Ala Ala 135 Ile met Gin Pro Gin 215 Gin Asn Leu met i2 0 Leu Asn Leu Giu Pro 200 Pro met Thr Leu 105 Ser Lys GlU ser Gin 185 Pro Ser Ala As n 90 Glu Pro His Leu Lys i7 0 Trp Gin Pro Met Asn 250 110 Tyr 75 Trp Arg Lye Ile Gin 155 Gin Asp Gin Phe Arg 235 Leu Ser met Aen Gin Glu Leu i2 Arg Ser 140 Arg Lys Ile Lye Giu Gin His Gin 205 Leu Asn 220 Arg Asn Giy Arg Giu Arg 110 Gin Arg Giu Giu Asn 190 Ile Met Asp Arg Ala Giu Arg Gin Tyr His Asn Lye Lys Arg 175 His Gin Gly Leu Cys 255 Leu Asn Tyr Leu Aen Ala 160 Glu Giy His Gly Asp 240 Val Tyr Asn Cys INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 1345 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 149. .968 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1. .i345 OTHER INFORMATION: /note= "product =zea mays APi."' (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GCACGAGTCc TCCTCCTCCT CGCATCCCAC CCCACCCCAC CTTCTCCTTA AAGCTACCTG *CCTACCCGGC GGTTGCGCGC CGCAATCGAT CGACCGGAAG AGAAAGAGCA GCTAGCTAGC TAGCAGATCG GAGCACGGCA ACAAGGCG ATG GGG CGC GGC AAG GTA CAG CTG Met Gly Arg Gly Lys Val Gin Leu 41

'I

WO 97/46079 PCTIUS97/09682 AAG C Lys

CG

Arg 2

GCCC

Ala C TAC C Tyr I

TAT

Tyr

GGA

Giy

ATA

Ile4 105

AAT

Asn

AAG

Lys

GAG

GiU

CAG

Gin

CAG

Gin 185

CAG

Gin

GGA

Gly

GAT

GG

Lrg

;AG

;cc la 3er

%AT

CAL

CCC

Pro

CAC

His

CTA

Leu

AAG

Lys 170

CA.A

Gin

ACA

Thxe

CTC

Leiu

.GJ

ATA

Ile

GGC

Gly

GTC

Val

ACC

Thr

TAT

Tyr 75

TGG

Trp

AAA

Lys

AAA

Lys

ATC

Ile

CAG

Gin 155

GAA

Giu

CAG

Gin

AGC

-ser

;CCG

iPro

SGO.]

C

;AG AAC AAG ;iu Aen Lys

ATA

Ile 15 AAC COG CAG OTO ACC TTC TCC AAG COC Asn Arg Gin Val Thr Phe ser Lye Arg

CTG

Leu

GCC

Ala

GAC

Asp 60

OCT

Al a

TOC

Cy s

TGC

Cys

GAG

Giu

AGA

Arg 140

AAG

Lys

CTT

Leu

CAG

Gin

TCA

Ser

CC]

Pro 220

GAP

oh,.

CTC AAG LeU Lye 30 GTC ATC Val Ile TCC CGC ser Arg GAA AAG4 Glu~ Lys CAC GAA His GIU CAC AAG His Lye 110 CTC CAG Leu Gin 125 TCA AGO Ser Arg AAG GAG Lye Giu GCO GAG Ala Glu CAG GTG Gin Val 190 TCA TCG Ser Ser 205 CCA CAC Pro His GAO CTO Giu Leu U. GOCG CAC GAG

I

-bye 3TC ,Jai M4et

GCT

Ala

TAC

Tyr 95

CAC

His

CAA

Oin

AAG

Lye

AGG

Arg

AGO

Arg 175

CAG

Gin

TCC

Ser

AAC

Asr GC9J Al~ Ala

TTC

Phe

GAC

Asp

CTT

Leu 80

AGO,

Arg

CTG

Leu

CTA

Leu

AGC

Ser

TCA

Ser 160

CAG

Gin

TOO

Trp

TCC

Ser

ATC

GC(

24( His C TCC C ser I

AAA

Lys 65

ATT

Ile

AAA(

Lys

ATO

Met

GAG

Oiu

CAC

His 145

CTG

Leu

AAG

Lye

KGAC

Asp

TTC

*Phe

TOC

Cys 225

CG

I Ala

;CAG

:i Gin liu cc ~ro 50

OT

Ilie VCA4 5er

:TG

Leu

GGA

Gly

CAG

Gin 130

CTT

Leu

CAG

Gin 0CC Ala

CAG

Gin

ATG

Met 210

TTC

Phe

GCG

Ala

CTC

Leu Tc Ile 35

AG

,ys4 CTT4 Lieu4

GCT

Ala Lays

GAG

Giu 115

CAG

Gin

ATG

Met

GAG

Giu

GTC

Val

CAG

Gin 195

ATG

met

CCG

Pro

GCG

rcc Ser

GGC

31y

GAA

G1U

GAA

Glu

GCC

Ala 100

GAT

Asp

CTG

Leu 0CC Ala

GAG

Glu

GCO

Al a 180

ACA~

Thr

AGG

Arg

CCC

Prc 0CC

GTC

Val

AAG

Lye

CGC

Arg TCT4 Ser

AAA.

Lye

CTA

Leu

GAT

Asp

GAG

Giu

AAC

As n 165

*AGC

*Ser

CAT

*His

CAG

Gin

TTO

Leu

;CAG

:TC

CTC

Lieu

TAT

ryr

GAA

GlU

ATT

Ile

GAG

Glu

AGC

Ser

TCT

Ser 150

AAG

Lys

CGG

Arg 0CC Ala

GAT]

Asp

ACA~

Thr 230

CAC

TGC OAT CyS Asp TAC GAG Tyr Glu GAO CGA GlU Arg ACT GAG Ser GlU GAO ACC Glu Thr TCT TTG Ser Leu 120 TCA CTO Ser Leu 135 ATT TCT Ile Ser OCT CTO Ala Leu CAG CAG Gln Gin CAG 0CC Gin Ala 200 CAG CAG Gin Gin 215 ATO GGA Met Gly CAG CAG Gin Oln CTG CCA 220 268 316 364 412 460 508 556 604 652 700 748 796 844 892 940 Asp Arg Oly 235 Ala Ala Gin Gir 245 CCA CTO CCO 000 CAG GCO CAA CCC Pro Leu Pro Oly Gin Ala Gin Prc 250 255 Arg Ile Ala Gly Leu Pro 260 CCA TGG Pro Trp 265 ATG CTG AGC CAC Met Leu Ser His 270 CTC AAT GCA Leu Asn Ala T AAGGAGAOGG TCGATOAACA 988

I,

WO 97/46079 PCT/US97/09682 112 CATCGACCTC CTCTCTCTCT CTCTCTCGTC ATGGATCATG ACGTACGCGT ACCATATGGT TGCTGTGCCT GCCCCCATCG ATCGCGAGCA ATGGCACGCT CATGCAAGTG ATCATTGCTC CCCGTTGGTT AAACCCTAGC CTATGTTCAT GGCGTCAGCA ACTAAGCTAA ACTATTGTTA TGTTTGCAAG AAAGGGTAAA CCCGCTAGCT GTGTAATCTT GTCCAGCTAT CAGTATGCTT GTTACTGCCC AGTTACCCTT GAATCTAGCG GCGCTTTTGG TGAGAGGGTG CAGTTTACTT TAAACATGGT TCGTGACTTG CTGTAAATAG TAGTATTAAT CGATTTGGGC ATCTAAA INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 273 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 1048 1108 1168 1228 1288 1345 Met Giy Arg Gly Lys Val Gin Leu Lys Arg Ile Giu Aen Lys Ile Aen krg Gin Val His GiU Ile Ser Pro Lye Lys Ile Leu Ile Set Ala Lys Leu Lye Met Giy Giu 115 Giu Gin Gin 130 His Leu met 145 Leu Gin Glu Lys Ala Val Asp Gin Gin 195 Thr Set Giy Giu Glu Ala 100 Asp Leu Al a GlU Ala 180 Thr Phe Val Lye Arg Ser Lye Leu Asp Giu As n 165 Ser Ser Leu Leu Tyr 70 GiU Ile GiU Ser Ser 150 Lye Arg Lys Cys Tyr 55 GiU Ser Giu Ser Set 135 Ile Ala Gin Arg Asp 40 Giu Arg Giu Thr Leu 120 Leu Ser Leu Gln Arg 25 Ala Tyr Tyr Gly Ile 105 Aen Lye GiU Gin Gin 185 As n Giu Ala Ser Asn 90 Gin Pro His Leu Lye 170 Gin Thr Gly Leu Vai Ala Thr Asp Tyr Ala 75 Trp Cys Lye Cys Lye Giu Ile Arg 140 Gin Lye 155 GlU LeU Gin Gin Ser Set Leu Val Set Giu His His Leu 125 Ser Lys Ala Gin Ser 205 Lys Ile Arg Lye Giu Lys 110 Gin Arg Glu Giu Vai 190 Set Lys Val Met Ala Tyr His Gin Lys Arg Arg 175 Gin Set Ala Phe Asp Leu Arg Leu LeU Set Set 160 Gin Trp Set His Ala Gin Ala Gin 200 Phe Met 210 PeMetMet Arg Gin Asp Gin Gin Gly Leu Pro Pro 210215 220 Pro His Aen Ile WO 97/46079 PCTUS97/09682 113 Cys 225 Phe Pro Pro Leu Thr Met Gly Asp Arg Gly 230 235 Gin Gin Gin Gin Pro Leu Pro 245 250 Glu Glu Leu Ala Ala 240 Ala Ala Ala Ala Gly Gin Ala Gin Pro 255 Gln Leu Arg Ile Ala Gly Leu Pro Pro Trp 260 265 Met Leu Ser His Leu Asn 270 Ala INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 779 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 10..775 (ix) FEATURE: NAME/KEY: unsure LOCATION: 778..779 OTHER INFORMATION: /note= nucleotides." (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1..779 OTHER INFORMATION: /note= thaliana CAL." "N one or more "product Arabidopsis (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: TTAAGAGAA ATG GGA AGG GGT AGG GTT GAA TTG AAG AGG ATA GAG AAC Met Gly Arg Gly Arg Val Glu Leu Lys Arg Ile Glu Asn 1 5 AAG ATC Lys Ile AAT AGA CAA GTG Asn Arg Gin Val TTC TCG AAA AGA Phe Ser Lys Arg AGA ACT GGT CTT TTG Arg Thr Gly Leu Leu GCC GAG GTT TCC CTT Ala Glu Val Ser Leu

AAG

Lys AAA GCT CAG GAG Lys Ala Gin Glu TCT GTT CTT TGT Ser Val Leu Cys

GAT

Asp ATT GTC TTC TCC Ile Val Phe Ser

CAT

His AAG GGC AAA TTG Lys Gly Lys Leu GAG TAC TCC TCT Glu Tyr Ser Ser GAA TCT Glu Ser TGC ATG GAG Cys Met Glu

AAG

Lys GTA CTA GAA CGC Val Leu Glu Arg

TAC

Tyr 70 GAG AGG TAT TCT Glu Arg Tyr Ser TAC GCC GAG 240 Tyr Ala Glu AGA CAG CTG ATT GCA CCT GAC TCT CAC GTT AAT GCA CAG Arg Gln Leu Ile Ala Pro Asp Ser His Val Asn Ala Gin 85 ACG AAC TGG Thr Asn Trp WO 97/46079 WO 9746079PCT1US97/09682

TCA

Ser

AAC

Asn 110

GAT

Asp

CGC

Arg

AGA

Arg

ATA

Ile

CAG

Gin 190

CAC

His

ATG

Met

AAT

As n

GCC

Ala

ATG

Met

CAA

Gin

CTC

Leu

TCC

Ser

AAG

Lys

AAG

Lys 175

CTG

Leu Ccc Pro

GGT

Giy

CTG

Leu

GCT

Aia 255 GAG TAT AGC AGG CTT AAG Giu Tyr Ser Arg Leu Lys 100 AGG CAT TAT CTG GGA GAA Arg His Tyr Leu Gly Giu 115 CAA AAT CTG GAG CAG CAG Gin Asn Leu Giu Gin Gin 130 AGA AAA AAT CAA CTC ATG Arg Lys Asn Gin Leu Met 145 GAG AAG GAG ATA CAG GAG Giu Lys Giu Ile Gin Giu 160 165 GAG AGG GAA AAC ATC CTA Giu Arg Giu An Ile Leu 180 AAC CGC AGC GTC GAC GAT Asn Arg Ser Val Asp Asp 195 CAT CTT TAC ATG ATC GCT His Leu Tyr Met Ile Ala 210 GGT TTG TAC CAA GGA GAA Giy Leu Tyr Gin Giy Giu 225 GAT CTG ACT CTT GAA CCC Asp Leu Thr Leu Glu Pro 240 245 T GANN 114 AAG ATT Lys Ile TTG GAA Leu Glu 120 GAG ACT Giu Thr 135 GAG TCC Giu Ser AAC AGC Aen Ser ACA AAA Thr Lys CCA CAG Pro Gin 200 CAG ACT Gin Thr 215 CAA ACG Gin Tkir

CTT

Leu

ATG

Met

CTT

Leu

AAC

Asn

CTT

Leu 170

ACC

Thr

CAA

Gin

CCT

Pro

ATG

Met

TTG

Leu

AGC

Ser

AAG

Lye

CAC

His 155

ACC

Thr

CAA

Gin

CCA

Pro

TTC

Phe

AGG

Arg 235

GAG

Giu

CTC

Lau

CAC

His 140

CTC

Leu

AAA

Lys

TGT

Cys

TTT

Phe

CTA

Leu 220

AGG

Arg

AGA

Arg

AAG

Lys 125

ATT

Ile

CAA

Gin

CAG

Gin

GAG

Giu

CAA

Gin 205

AAT

As n

AAC

Asn 336 384 432 480 528 624 ATT TAC AAT TAC CTT GGC TGT TAC Ile Tyr Asn Tyr Leu 250 Gly Cys Tyr INFORMATION FOR SEQ ID Met 1 Arg Gin Ser SEQUENCE CHAR~ACTERISTICS: LENGTH: 255 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE.: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1O: Giy Arg Giy Arg Val GiU Led Lye Arg Ile Giu Asn Lys Ile Aen 5 10 Gin Val Thr Phe Ser Lys Arg Arg Thr Giy Leu Leu Lys Lye Aia 25 Giu le Ser Vai Lau Cys Asp Ala Giu Val Ser Leu Ile Val Phe 40 His Lys Gly Lye Leu Phe Giu Tyr Ser Ser GiU Ser Cys met Giu 55

I,

WO 97/46079 PCTJUS97/09682 Lys Ile Tyr His As n Lys 145 Lys Arg Arg Leu Leu 225 Leu Val Ala Ser Tyr Leu 130 Asn Giu Giu Ser Tyr 210 Tyr Thr Leu Pro Arg Leu 115

GIU

Gin Ile As n Val 195 met Gin Leu GiU Asp Leu.

100 Gly Gin Leu Gin Ile 180 Asp Ile Giy Glu Tyr 70 His Ala Giu Leu Asn 150 Giu Lys Val His Asp 230 Ile Giu Arg Tyr Ser Asn Ile Giu 120 Thr Ser ser Lys Gin 200 Thr Thr Asn Ala Gin 105 Pro Aia Leu met Gin 185 Pro Ser Ala Tyr 115 Tyr 75 Thr Leu Ser Lys His 155 Thr Gin Pro Phe Arg 235 Gly Ala As n Giu Len His 140 Len Lys Cys Phe Leu 220 Arg Cys Giu Trp Arg Lys 125 Ile Gin Gin Gin Gin 205 Asn As n Tyr Arg Ser Asn 110 Asp Arg Arg Ile Gin 190 His met Aen Ala Leu Gin Arg Gin Arg Gin 160 Giu Aen His Giy Asp 240 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 756 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iX) FEATURE: NAME/KEY: CDS LOCATION: 1. .754 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1. .7596 OTHER INFORMATION: /note= "product =Brassica oieracea

CAL."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: ATG GGA AGG GGT AGG GTT GAA ATG AAG, AGG ATA GAG AAC AAG ATC AAC Met Gly Arg Gly Arg Val Gin Met Lys Arg Ile Gin Asn Lys Ile Aen 1 5 10 CGA CAA GTG ACG TTT TCG AAA AGA AGA GCT GGT CTT TTG AAG AAA GCC Arg Gin Val Thr Phe Ser Lys Arg Arg Aia Gly Leu Len Lys Lye Ala 25 WO 97/46079 WO 9746079PCTfJS97/09682 116 CAT GAG ATC His Giu Ile TCG ATC CTT TGT Ser Ile Len Cys

CAT

Asp 40 GCT GAG GTT TCC Ala Giu Val Ser

CTT

Len ATT GTC TTC Ile Val Phe 144 TCC CAT Ser His AAG GGG AAA CTG Lys Gly Lys Len GAG TAC TCG TCT Giu Tyr ser Ser TCT TGC ATG GAG Ser Cys met Giu

AAG

Lys GTA CTA GAA CAC Val Len Gin His

TAC

Tyr 70 GAG AGG TAC TCT Giu Arg Tyr Ser GCC GAG AAA CAG Ala Gin Lys Gin

CTA

Len 240 288 AAA OTT CCA GAC TCT CAC GTC AAT GCA CAA ACG AAC TGG TCA GTG GAA Lys Val Pro Asp Ser His Val Asn Ala Gin Thr Asn Trp Ser Vai Gin 90 TAT AGC AGG Tyr Her Arg CAT TAT CTG His Tyr Leu 115

CTT

Len 100 AAG OCT AAG ATT Lys Ala Lye Ile

GAG

Gin 105 CTT TTG GAG AGA Leu Leu Gin Arg AAC CAA AGG Aen Gin Arg 110 GAG CTA CAG Giu Len Gin GOC GAA GAT TTA Giy Giu Asp Leu

GAA

Giu 120 TCA ATC AGC ATA Ser Ile Her Ile

AAG

Lys 125 AAT CTG Asn Leu 130 GAG CAG CAG CTT Gin Gin Gin Len ACT TCT CTT AAA Thr Her Len Lys

CAT

His 140 ATT CGC TCG AGA Ile Arg Ser Arg

AAA

Lys 145 AAT CAA CTA ATG Asn Gin Len Met GAG TCC CTC AAC Giu Her Leu Asn

CAC

His 155 CTC CAA AGA AAG Len Gin Arg Lys AAA GAA ATA CTG Lye Gin Ile Len GAA AAC AGC, ATG Gin Asn Her Met

CTT

Len 170 GCC AAA CAG ATA Ala Lye Gin Ile AGO GAG Arg Gin 175 AGO GAG AGT Arg Gin Her CGC AGC CAC Arg Her His 195

ATC

Ile 180 CTA AGG ACA CAT Leu Arg Thr His

CAA

Gin 185 AAC CAA TCA GAG Asn Gin Her Giu CAG CAA AAC Gin Gin Aen 190 AAT CCT TAC Asn Pro Tyr 528 576 624 CAT GTA OCT CCT His Val Ala Pro CCG CAA CCG CAG Pro Gin Pro Gin

TTA

Len 205 ATO GCA Met Ala 210 TCA TCT CCT TTC Her Her Pro Phe AAT ATG GOT GGC Asn Met Gly Gly ATG TAC CAA GGA Met Ty Gin Gly 220 CTG ACT CTT OAR Len Thr Leu Gin

GAA

Gin

CCC

Pro 240

TAT

Tyr 225 CCA ACG GCG GTG Pro Thr Ala Val

AGG

Arg 230 AGO AAC COT CTC Arg Asn Arg Len

GAT

Asp 235 ATT TAC AAC TGC Ile Tyr Asn Cys CTT GGT TAC TTT Len Gly Tyr Phe GCC OCA T GA Ala Ala 250 INFOR.MATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 251 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein WO 97/46079 WO 9746079PCTIUS97/09682 117 NO: 12: (Xi) SEQUENCE DESCRIPTION: SEQ ID Met Gly Arg Giy Arg His Ser Lye Lye Tyr His As n Lys 145 Lye Arg Ar g met Tyr 225 Ile Gin Gin His Val Val Ser Tyr Leu 130 Asn Gin Gin Ser Ala 210 Pro Tyr Val Ile Lye Leu Pro Arg Leu 115 Gin Gin Ile Her His 195 Ser Thr Asn Thr Ser Gly Gin Asp Leu 100 Gly Gin Leu Len Ile 180 His ser Ala Cys Arg Phe Ile Lys His Ser Lye Giu Gin Met Gin 165 Len Val Pro Val Aen 245 Val Giu Met Lye Ser Len Len Tyr 70 His Ala Asp Leu His 150 Giu Arg Ala Phe Arg 230 Leu Lye Cys Phe 55 Gin Val Lys Len Asp 135 Gin Asn Thr Pro Len 215 Arg Gly Arg Asp 40 Giu Arg As n Ile Glu 120 Thr ser Her His Gin 200 Aen Asn Tyr Arg 25 Ala Tyr Tyr Ala Giu 105 Her ser Len Met Gin 185 Pro Met Arg Phe Arg Ile 10 Ala Gly Gin Val Her Her Her Tyr 75 Gin Thr 90 Leu Len Ile ser Leu Lye Asn His 155 Leu Ala 170 Aen Gin Gin Pro Giy Gly Len Asp 235 Ala Ala 250 Gin Len Ser Gin Ala Asn Gin Ile His 140 Leu Lys Her Gin Met 220 Len Aen Len Len Ser Gin Trp Arg Lye 125 Ile Gin Gin Gin Len 205 Tyr Thr Lys Lye Ile Cys Lys Her As n 110 Giu Arg Arg Ile Gin 190 Asn Gin Len Ile Lye Val met Gin Val Gin Len Ser Lyes Arg 175 Gin Pro Gly Gin As n Ala Phe Gin Len Gin Arg Gin Arg Gin 160 Gin Aen Tyr Gin Pro 240 INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 756 base pairs TYPE: nucleic acid STRANDEDNESH: donble TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .451 WO 97/46079 WO 9746079PCTIUS97/09682 118 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1. .7*96 OTHER INFORMATION: /note= "Product Brassica oieracea var. botrytis CAL.11 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: ATG GGA AGG GGT AGG GTT GAA ATG AAG AGG ATA GAG AAC AAG ATC AAC Met 1

AGA

Arg

CAT

His

TCC

Ser

AAG

Lys

AAA

Lys

TAT

Tyr

CAT

His

AAT

Asa

AAA

Lys 145 Gly Arg Gly

CAA

Gin

GAG

Giu

CAT

His

GTA

Val.

GCT

Ala

AGC

Ser

TAT

Tyr

CTG

Leu 130

AAT

GTG

Val

ATC

Ile

AAG

Lys

CTA

Leu

CCA

Pro

AGG

Arg

CTG

Lau 115

GAG

Giu

CAA

ACG

Thr

TCG

Ser

GGG

Gly

GAA

Giu

GAC

Asp

CTT

Leu 100

GGA

Giy

CAG

Gin

CTA

Arg 5

TTT

Phe

ATT

Ile

AA

Lye

CGC

Arg

TCT

Ser

AAG

Lys

GAA

GiU

CAG

Gin

ATG

Vai Giu Met Lys TCG AAA Ser Lye CTT TGT Leu Cys CTG TTC Leu Phe 55 TAC GAG Tyr Giu 70 CAC GTC His Val GCT AAG Aia Lys GAT TTA Asp Leu CTT GAC Leu Asp 135 CAC T A( His 150

AGA

Arg

GAT

Asp 40

GAG

GiU

AGG

Arg

AAT

Asan

ATT

Ile

GAA

Giu 120

ACT

AGA

Arg 25

GCT

Aia

TAC

Tyr

TAC

Tyr

GCA

Ala

GAG

Giu 105

TCA

Ser

TCT

Arg 10

GCT

Ala

GAG

GIU

TCG

Ser

TCT

Ser

CAA

Gin 90

CTT

Leu

ATC

Ile

CTT

Ile Giu Aen Lys Ile Aen

GGT

Gly

GTT

Val

TCT

Ser

TAC

Tyr 75

ACG

Thr

TGG

Trp

AGC

Ser

CTT

Lau

TCC

Ser

GAA

GiU 60

GCC

Ala

AAC

Asn

GAG

Giu

ATA

Ile

CAT

TTG

Leu

CTT

Lau 45

TCT

Ser

GAG

Giu

TGG

Trp

AGG

Arg

AAG

Lye 125

ATT

Ile

AAG

Lys 30

ATT

Ile

TGC

Cys

AAA

Lye

TCA

Ser

AAC

Aen 110

GAG

Giu

CGC

Arg

AAA

Lys

GTC

Vai

ATG

Met

CAG

Gin

ATG

Met

CAA

Gin

CTA

Leu

TCC

ser

GCC

Ala

TTC

Phe

GAG

Giu

CTA

Leu

GAA

GiU

AGG

Arg

CAG

Gin

AGA

Arg Thr Ser Leu Lys His ;TCCCTCAA CCACCTCCAA AGAAAGGAGA Asa Gin Leu Met

AAGAAATACT

TAAGGACACA

CGCAACCGCA

ACCAAGGAGA

TTTACAACTG

GGAGGAAAAC AGCATGCTTG CCAAACAGAT AAAGGAGAGG GAGAGTATCC TCAAAACCAA~ TCAGAGCAGC AAAACCGCAG CCACCATGTA GCTCCTCAGC GTTAAATCCT TACATGGCAT CATCTCCTTT CCTAAATAT'G GGTGGCATGT ATATCCAACG GCGGTGAGGA GGAACCGTCT CGATCTGACT CTTGAACCCA CAACCTTGGT TACTTTGCCG CATGA 481 541 601 661 721 756 WO 97/46079 PCT/US97/09682 119 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 150 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE Met Gly Arg Gly Arg Arg His Ser Lys Lys Tyr His Asn Lys 145 Gin Glu His Val Ala Ser Tyr Leu 130 Val Thr Ile Ser Lys Gly Leu Glu Pro Asp Arg Leu 100 Leu Gly 115 Glu Gin DESCRIPTION: SEQ ID Val Glu Met Lys Arg Ser Lys Arg Arg Ala 25 Leu Cys Asp Ala Glu 40 Leu Phe Glu Tyr Ser 55 Tyr Glu Arg Tyr Ser 70 His Val Asn Ala Gin Ala Lys Ile Glu Leu 105 Asp Leu Glu Ser Ile 120 Leu Asp Thr ser Leu 135 His 150 NO:14: Ile Glu Gly Leu Val Ser ser Glu Tyr Ala 75 Thr Asn Trp Glu Ser Ile Lys His 140 Asn Lys Leu Lys Leu Ile Ser Cys Glu Lys Trp Ser Arg Asn 110 Lys Glu 125 Ile Arg Ile Lys Val Met Gin Met Gin Leu Ser Asn Ala Phe Glu Leu Glu Arg Gin Arg Asn Gin Leu Met INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1500 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 72..1343 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1..1500 OTHER INFORMATION: /note= "product Arabidopsis thaliana LEAFY (LFY)." (xi) SEQUENCE DESCRIPTION: SEQ ID AAAGCAATCT GCTCAAAAGA GTAAAGAAAG AGAGAAAAAG AGAGTGATAG AGAGAGAGAG WO 97/46079 WO 9746079PCTIUS97/09682 120 AAAAATAGAT T ATG GAT CCT GA&. GGT TTC ACG AGT GGC TTA TTC CGG TGG Met Asp Pro Giu Gly Phe Thr ser Gly Leu Phe Arg Trp 1 5

AAC

Asn

CTG

Leu

CTT

Leu

ACG

Thr

GGT

Gly

ATC

I le

GCC

Ala 110

TCT

Ser

ACT

Thr

TTA

Leu

AAT

Asn

ATG

Met 190

TCA

Ser

ATG

Met

GAG

Glu

CCA

Pro 1s

CAG

Gin

GGT

Gly

GCG

Ala

ATG

Met

TTT

Phe

GTT

Val

TCT

Ser

CAT

His

TCT

Ser

AAC

Asn 175

AAG

Lys

GTG

Val

GAT

Asp

CAT

His

ACG

Thr

CAA

Gin

GGT

Gly

GCG

Ala

AAG

Ly s

CGT

Arg

AGA

Arg

AGA

Arg

CAC

His

GAG

Glu 160

GGC

Gly

AAG,

Lys

GAA

Glu

AAC

As n

CCG

Pro 240 AGA GCA TTG GTT CAA GCA CCA CCT CCG GTT CCA CCT CCG Arg

CAG

Gin

TTA

Leu

AAG

Lys

GAC

Asp

TGG

Trp

GCT

Ala

CGC

Arg

GCT

Ala 145

GAA

Giu

GGA

Gly

CAA

Gin

ACC

Thr

GGC

Gly 225

TTT

Phe Ala

CCG

Pro

GAG

GlU

ATA

Ile

GAG

Giu

GAG

Giu

GAA

GiU

CGT

Axg 130

CTT

Leu

CCG

Pro

GGA

Giy

CAG

Gin

GAC

Asp 210

AAC

Asn

ATC

Ile Leu

GTG

Val 35

GGA

Giy

GCG

Ala

GAG

GiU

CTT

Leu

CGG

Arg 115

CAT

His

GAT

Asp

GTG

Val

GGA

Gly

CAG

Gin 195

GAA

Glu

GGA

Gly

GTA

Val Val 20

ACA

Thr

CTA

Leu

GAG

Giu

CTT

Leu

CTT

Leu 100

AGA

Arg

TTG

Leu

GCT

Ala

CAG

Gin

AGT

Ser 180

CAG

Gin

GAC

Asp

GGT

Gly

ACG

Thr Gin

CCG

Pro

TTC

Phe

TTA

Leu

GAA

GiU 85

GTT

Val

CGA

Arg

CTA

Leu

CTC

Leu

CAA

Gin 165

GGT

Gly

AGA

Arg

GTC

Val

AGT

Ser

GAG

Giu 245 Ala

CAG

Gin

GGT

Gly

GGT

Gly 70

GAG

Giu

GGT

Gly

TTG

Leu

CTC

Leu

TCC

Ser 150

CAA

Gin

TAC

Tyr

CGG

Arg

AAC

Asn

GGT

Gly 230

CCT

Pro Pro

ACG

Thr

CCA

Pro 55

TTT

Phe

ATG

met

GAA

Giu

CAA

Gin

TCC

Ser 135

CAA

Gin

GAC

Asp

TGG

Trp

AGA

Arg

GAA

Giu 215

TTG

Leu

GGG

Gly Pro

GCT

Ala 40

TAC

Tyr

ACG

Thr

ATG

Met

CGG

Arg

GAA

GiU 120

GCC

Ala

GAA

Giu

CAG

Gin

GAC

Asp

AAG

Lye 200

GGT

Gly

GGG

Gly

GAA

Giu Pro

GCT

Ala

GGT

Gly

GCG

Ala

AAT

Asn

TAC

Tyr 105

GAG

Glu

GCT

Ala

GAT

Asp

ACT

Thr

GCA

Ala 185

AAA

Lys

GAG

Glu

ACA

Thr

GTG

Val Val

TTT

Phe

ATA

Ile

AGC

Ser

AGT

ser

GT

Gly

GAG

Giu

GGT

Gly

GAT

Asp

GAT

Asp 170

GGT

Gly

CCA

Pro

GAT

Asp

GAG

GiU

GCA

Ala 250 Pro

GG

Gly

CGT

Arg

ACG

Thr

CTC

Leu

ATC

Ile

GAA

GiU

GAT

Asp

TGG

Trp 155

GCG

Ala

CAA

Gin

ATG

met

GAC

Asp

AGA

Arg 235

COT

Arg Pro

ATG

Met

TTC

Phe

CTT

LeU

TCT

Ser

AAA

Lye

GAG

Giu

TCC

Ser 140

ACA

Thr

GCG

Ala

GA

Gly

CTG

Leu

GAC

Asp 220

CAG

Gin

GGC

Gly Pro

CGA

Arg

TAC

Tyr

GTG

Val

CAT

His

GCT

Ala

GAA

GiU 125

GGT

Gly

GG

Gly

GGG

Gly

AAG

Lys

ACG

Thr 205

GGG

Gly

AGG

Arg

AAA

Lys 206 254 302 350 398 446 494 AAG AAC Lys Asn 255 GGC TTA GAT TAT CTG TTC CAC TTG TAC GAA CAA TGC CGT GAG Gly LeU Asp Tyr Phe His LeU Tyr GlU Gin Cys Arg Glu WO 97/46079 WO 9746079PCT/US97/09682

TTC

Phe 270

CCC

Pro

GCG

Ala

GCT

Ala

TTT

Phe

AAG

Lys 350

GTC

Val

CTG

Leu

GCG

Ala

CGT

CTT

LeU

ACC

Thr

AGT

Ser

CTC

Leu

AAA

Lys 335

CCA

Pro

TTT

Phe

CGT

Arg

GCT

Ala

GGT

CTT

Leu

AAG

Ly s

TAC

Tyr

CAC

His 320

GAA

Giu

CTT

Leu

AAC

As n

CAG

Gin

TTA

Leu 400

GGA

CAG

Gin

GTG

Val

ATA

Ile 305

TGC

Cys

CGC

Arg

GTG

Val

GCT

Ala

CTT

Leu 385

GTT

Val

TGC

GTC

Val

ACG

Thr 290

AAC

Asn

CTA

Leu

GGT

Gly

AAC

Asn

CAT

His 370

TGC

Cy 5

GGC

Gly

GGC

CAG

Gin 275

AAC

Asn

AAG

Lys

GAC

Asp

GAG

GlU

ATC

Ile 355

CCT

Pro

CAT

His

GGT

Gly

GGC

ACA

Thr

CAA

Gin

CCT

Pro

GAA

GiU

AAC

Asn 340 Ala

CGT

Arg

TTG

Leu

ATT

Ile

GAC

ATT

Ile

OTA

Val

AAA

Lys

GAA

GlU 325

GTT

Val

TGT

Cys

CTC

Leu

GAG

Giu

AGC

Ser 405

GAC

GCT

Ala

TTC

Phe

ATG

Met 310

GCT

Ala

GGC

Gly

CGT

Arg

TCT

Ser

CGG

Arg 390

TGT

Cys

TTG

121 AAA GAC Lys Asp 280 AGG TAC Arg Tyr 295 CGA CAC Arg His TCA AAT Ser Asn TCA TGG Ser Trp CAT GGC His Gly 360 ATT TGG Ile Trp 375 A-AC AAT Aen Asn ACC GGA Thr Gly CGT TTC

CGT

Arg

GCG

Ala

TAC

Tyr

GCT

Ala

CGT

Arg 345

TGG

Trp

TAT

Tyr

GCG

Ala

TCG

GGC

Gly

AAG

Lys

GTT

Val

CTC

Leu 330

CAG

Gin

GAT

Asp

GTT

Val

GTT

Val

TCG

GAA

Glu

AAA

Lys

CAC

His 315

AGA

Arg

GCT

Ala

ATA

Ile

CCA

Pro

GCT

Ala 395

ACG

AAA

Lys

TCA

Ser 300

TGT

Cys

AGA

Arg

TGT

Cys

GAC

Asp

ACA

Thr 380

GCG

Ala

TCT

TGC

Cys 285

GGA

Gly

TAC

Tyr

GCG

Ala

TAC

Tyr

GCC

Ala 3615

AAG

Lys

GCT

Ala

GGA

Gly 926 974 1022 1070 1118 1166 1214 1262 1310 1363 1423 1483 1500 Ser Ser Thr ser TAGTTTGGTT TGGGTAGTTG Arg Gly Giy Cys Gly Gly Asp Asp 415 420 Leu Arg Phe TGGTTTGTTT AGTCGTTATC CTAATTAACT ATTAGTCTTT AATTTAGTCT TCTTGGCTAA TTTATTTTTC TTTTTTTGTC AAAACCTTTA ATTTGTTATG GCTAATTTGT TATACACGCA GTTTTCTTAA~ TGCGTTA INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 424 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Met Asp Pro Glu Gly Phe Thr Ser Gly Leu Phe Arg Trp A-sn Pro Thr 1 5 10 Arg Ala Leu Val Gin Ala Pro Pro Pro Val Pro Pro Pro Leu Gin Gin 25 Gin Pro Val Thr Pro Gin Thr Ala Ala Phe Giy Met Arg Leu Gly Gly 40 WO 97/46079 PCTIUS97/09682 Len I Lys Asp Trp Ala Arg Ala 145 Glu Gly Gln Thr Gly 225 Phe Len Gin Val Ile 305 Cys Arg Val Ala Leu 385 lu Ile Gl Glu Glu Arg 130 Len Pro Gly Gin Asp 210 Asn Ile Asp Val Thr 290 Asn Len Gly Asn His 370 cys Gly I Ala C Glu Leu Arg 115 His Asp Val Gly Gin 195 Glu Gly Val Tyr Gin 275 Asn Lys Asp Glu Ile 355 Pro His .eu Phe Gly ;lu eu :,eu 100 .rg Leu Ala Gin Ser 180 Gln Asp Gly Thr Leu 260 Thr Gin Pro Glu Asn 340 Ala Arg Leu Leu Glu Val Arg Leu Leu Gin 165 Gly Arg Val Ser Gl 245 Phe Ile Val Lye Gl 325 Val cys Leu Glu ;ly 70 3lu 31y Leu Len ser 150 Gin Tyr Arg Asn Gly 230 Pro His Ala Phe Met 310 Ala Gly Arg Ser Arg 390 122 Pro Tyr Gly Ile Arg 55 Phe Thr Ala Ser Thr 75 Met Met Asn Ser Leu 90 Glu Arg Tyr Gly Ile 105 Gin Glu Glu Glu Glu 120 Ser Ala Ala Gly Asp 135 Gin Glu Asp Asp Trp 155 Asp Gin Thr Asp Ala 170 Trp Asp Ala Gly Gin 185 Arg Lys Lys Pro Met 200 Glu Gly Glu Asp Asp 215 Leu Gly Thr Glu Arg 235 Gly Giu Val Ala Arg 250 Leu Tyr Giu Gin Cys 265 Lys Asp Arg Gly Glu 280 Arg Tyr Ala Lye Lys 295 Arg His Tyr Val His 315 Ser Asn Ala Leu Arg 330 Ser Trp Arg Gin Ala 345 His Gly Trp Asp Ile 360 Ile Trp Tyr Val Pro 375 F Asn Asn Ala Val Ala 395 Phe Leu Ser Lys Glu Ser 140 Thr Ala Gly Len Asp 220 Gln Gly Arg Lys ser 300 Cys Arg Cys Asp Thr 380 Ala Tyr Val His Ala Glu 125 Gly Gly Gly Lys Thr 205 Gly Arg Lys Glu Cys 285 Gly Tyr Ala Tyr Ala 365 Lys Ala Thr Gly Ile Ala 110 Ser Thr Leu Asn Met 190 Ser Met Glu Lys Phe 270 Pro Ala Ala Phe Lys 350 Val Leu Ala Ala Met Phe Val.

Ser His Ser Asn 175 Lye Val Asp His Asn 255 Len Thr Ser Len Lys 335 Pro Phe Arg Ala Ala Lys Arg Arg Arg His Glu 160 Gly Lye Gl Asn Pro 240 Gly Leu Lys Tyr His 320 Glu Len Asn Gln Len 400 WO 97/46079 PCT/US97/09682 123 Val Gly Gly Ile Ser Cys Thr Gly Ser Ser 405 410 Thr Ser Gly Arg Gly Gly 415 Cys Gly Gly Asp Asp Leu Arg Phe 420 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 1656 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1651 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1..1656 OTHER INFORMATION: /note= "domain ecdysone receptor ligand binding domain." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: ATG CGG CCG GAA TGC GTC GTC CCG GAG AAC CAA TGT GCG ATG AAG CGG Met Arg Pro Glu Cys Val Val Pro Glu Asn Gin Cys Ala Met Lys Arg 1 5 10 CGC GAA AAG Arg Glu Lys AGC TCT CAG Ser Ser Gin GCC CAG AAG GAG Ala Gin Lys Glu

AAG

Lys GAC AAA ATG ACC Asp Lys Met Thr ACT TCG CCG Thr Ser Pro GGC GGC CAA Gly Gly Gln CAT GGC GGC AAT His Gly Gly Asn

GGC

Gly AGC TTG GCC TCT Ser Leu Ala Ser GAC TTT Asp Phe GTT AAG AAG GAG Val Lys Lys Glu

ATT

Ile CTT GAC CTT ATG Leu Asp Leu Met TGC GAG CCG CCC Cys Glu Pro Pro

CAG

Gin CAT GCC ACT ATT His Ala Thr Ile CTA CTA CCT GAT Leu Leu Pro Asp

GAA

Glu ATA TTG GCC AAG Ile Leu Ala Lys CAA GCG CGC AAT Gin Ala Arg Asn CCT TCC TTA ACG Pro Ser Leu Thr AAT CAG TTG GCC Asn Gin Leu Ala GTT ATA Val Ile 144 192 240 288 336 384 432 480 TAC AAG TTA Tyr Lys Leu GAT CTC AGG Asp Leu Arg 115

ATT

Ile 100 TGG TAC CAG GAT Trp Tyr Gin Asp

GGC

Gly 105 TAT GAG CAG CCA Tyr Glu Gin Pro TCT GAA GAG Ser Glu Glu 110 AGC CAA ACG Ser Gin Thr CGT ATA ATG AGT Arg Ile Met Ser

CAA

Gln 120 CCC GAT GAG AAC Pro Asp Glu Asn

GAG

Glu 125 GAC GTC Asp Val 130 AGC TTT CGG CAT Ser Phe Arg His ACC GAG ATA ACC Thr Glu Ile Thr

ATA

Ile 140 CTC ACG GTC CAG Leu Thr Val Gin

TTG

Leu 145 ATT GTT GAG TTT Ile Val Glu Phe

GCT

Ala 150 AAA GGT CTA CCA Lys Gly Leu Pro

GCG

Ala 155 TTT ACA AAG ATA Phe Thr Lys Ile

CCC

Pro 160 WO 97/46079 WO 9746079PCTIUS97/09682 124

CAGC

GinC

ATG

met

TTC

Phe2

ATG

Met

TCG

ser 225

ATC

Ile

ATC

Ile

CAC

His

ATC

Ile

TTC

Phe 305

ATC

Ile

ATT

Ile

GCA

Aia

TCC

Ser

CCC

Pro 385

ACA

Thr wAG GAC CAG ATC ACG TTA CTA AAG GCC liU Asp Gin Ile Thr Leu Leu Lys Ala 165 170

TGC

:TG

.jeu

XCG

11a

XCT

kia 210 M4et

TC

Phe

CAG

Gin

TGC

Cy s

CTC

Leu 290

TCA

Ser

TGG

Trp

ACC

Thr

TCG

Ser

ACT

Thr 370

CAG

Gin

CAG

Gin CGT2 Arg AAT2 Asn 195

GAT

Asp

AAG

Lys

TCG

Ser

AGC

Ser

GGC

Giy 275

ACC

Thr

CTA

Leu

GAC

Asp

CAG

Gin

GTT

Val 355

TCG

Ser ccc Pro

CCG

Pro kTG 4et 180

U.T

ksn

NAC

k.sn

GTG

Vl

GAC

Asp

TAC

Tyr 260

GAC

Asp

GAG

Giu

AAG

Ly s

GTT

Vai

GAG

Giu 340

GGG

Gly

GCG

Aia

CAA

Gin

CAG

Gin

GCA

Aia

AGA

Arg

ATT

Ile

GAC

Asp

CGG

Arg 245

TAC

Tyr

TCA

Ser

CTG

Leu

CTC

Leu

CAT

His 325

GAG

GiU

GGC

Giy

GCG

Aia

CCC

Pro

ICTA

Leu 405

:GA

:krg

NCA

Ser

GA.A

Giu

AAC

pAsn 230

CCG

Pro

ATC

Ile

ATG

met

CGT

Arg

AAA

Lys 310

GCC

Aia

AAC

Asn

GCC

Ala

GCA

Aia

TCC

Ser 390

CAA~

Gin

CGC

Arg

TAT

Tyr

GAC

Asp 215

GTC

Val

GC

Gly

GAC

Asp

AGC

Ser

ACG

Thr 295

AAC

As n

ATC

Ile

GAG

Glu

ATT

Ile

GCC

Al a 375

TCC

Ser

CCT

Pro

TAT

Tyr

ACG

Thr 200

CTG

Leu

GAA

Giu

CTG

Leu

ACG

Thr

CTC

Leu 280

CTG

Leu

CGC

Arg

CCG

Pro

CGT

Arg

ACC

Thr 360

GCG

Ala

CTG

Leu

CAG

Gin

GAC

Asp 185

CGG

Arg

CTG

Leu

TAC

Tyr

GAG

Giu

CTA

Leu 265

GTC

Val

GGC

Gly

AAA

Lys

CCA

Pro

CTC

Leu 345

GCC

Ala

GCC

Ala

ACC

Thr

CTA

Leu

CAC

His

GAT

Asp

CAT

His

GCG

Ala*

AAG

Ly s 250

CC

Arg

TTC

Phe

AAC

As n

CTG

Leti

TCG

Ser 330

GAG

Glu

GCC

Gly

CAG

Gin

CAG

Gin

CCA

Pro 410 Cys PiGC Ser

TCT

Ser

TTC

Phe

CTT

Leu 235

GCC

Ala

ATT

Ile

TAC

Tyr

CAG

Gin

CCC

Pro 315

GTC

Val

CGG

Arg

ATTI

Ile

CATI

His

AAC

Asr 395 CC I Prc

TCG

Ser

TCG

Ser

TAC

Tyr

TGC

Cys 220

CTC

Leu

CAA

Gin

TAT

Tyr

GCA

Ala

AAC

Asn 300

AAG

Lys

CAG

Gin

GCT

Ala

GAT

Asp

CAG

Gin 380

GAT

Asp

CAG

Gin rCG Ser 3AC Usp

L.AA

Lys 205

CGC

Arg

ACT

Thr

CTA

Leu

ATA

Ile

AAG

Lys 285

GCC

Ala

TTC

Phe

TCG

Ser

GAG

Ciu

TGC

Cys 365

CCT

Pro

TCC

Ser

CTG

Leu

GAG

Glu

TCA

Ser 190

ATG

Met

CAA

Gin

GCC

Ala

GTC

Val

CTC

Leu 270

CTG

Leu

GAG

ClU

CTC

Leu

CAC

His

CGT

Arg 350

GAC

Asp

CAG

Gin

CAG

Gin

CAA

Gin

GTG

Val 175

ATA

Ile

GCC

Ala

ATG

Met

ATT

Ile

GAA

GiU 255

AAC

As n

CTC

Leu

ATG

met

GAG

Glu

CTT

Leu 335

ATG

met

TCT

ser

CCT

Pro

CAC

His

GGTI

Gili 415

ATG

met

TTC

Phe

GGA

Cly

TTC

Phe

GTG

Val 240

GCG

Ala

CGC

Arg

TCG

Ser

TGT

Cys

GAG

GilU 320

CAG

Gin

CGG

Arg dCC Ala

CAG

Gin

CAG

Gin 400

CAA

Gin 528 576 624 672 720 768 816 864 912 960 1008 1056 1104 1152 1200 1248 1296 CTG CAA CCC CAG CTC CAA CCA CAG CTT CAG ACG CAA CTC CAG CCA CAG Leu Gin Pro Leu Gin Pro Gin Gin Thr Gin Leu Gin Pro Gin 430 WO 97/46079 PCT/US97/09682 125 ATT CAA CCA CAG CCA CAG CTC CTT CCC GTC TCC GCT CCC GTG CCC GCC 1344 Ile Gln Pro Gin Pro Gin Leu Len Pro Val Ser Ala Pro Val Pro Ala 435 440 445 TCC GTA ACC GCA CCT GGT TCC TTG TCC GCG GTC AGT ACG AGC AGC GAA 1392 Ser Val Thr Ala Pro Gly ser Leu Ser Ala Val Ser Thr Ser Ser Glu 450 455 460 TAC ATG GGC GGA AGT GCG GCC ATA GGA CCC ATC ACG CCG GCA ACC ACC 1440 Tyr Met Gly Gly ser Ala Ala Ile Gly Pro Ile Thr Pro Ala Thr Thr 465 470 475 480 AGC AGT ATC ACG GCT GCC GTT ACC GCT AGC TCC ACC ACA TCA GCG GTA 1488 Ser Ser Ile Thr Ala Ala Val Thr Ala Ser Ser Thr Thr ser Ala Val 485 490 495 CCG ATG GGC AAC GGA GTT GGA GTC GGT GTT GGG GTG GGC GGC AAC GTC 1536 Pro Met Gly Asn Gly Val Gly Val Gly Val Gly Val Gly Gly Asn Val 500 505 510 AGC ATG TAT GCG AAC GCC CAG ACG GCG ATG GCC TTG ATG GGT GTA GCC 1584 Ser Met Tyr Ala Asn Ala Gin Thr Ala Met Ala Leu Met Gly Val Ala 515 520 525 CTG CAT TCG CAC CAA GAG CAG CTT ATC GGG GGA GTG GCG GTT AAG TCG 1632 Leu His Ser His Gin Glu Gin Leu Ile Gly Gly Val Ala Val Lys Ser 530 535 540 GAG CAC TCG ACG ACT GCA T AGCAG 1656 Glu His Ser Thr Thr Ala 545 550 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 550 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: Met Arg Pro Glu Cys Val Val Pro Glu Asn Gin Cys Ala Met Lys Arg 1 5 10 Arg Glu Lys Lys Ala Gin Lys Glu Lys Asp Lys Met Thr Thr ser Pro 25 Ser Ser Gin His Gly Gly Asn Gly Ser Leu Ala Ser Gly Gly Gly Gin 40 Asp Phe Val Lys Lys Glu Ile Leu Asp Leu Met Thr Cys Glu Pro Pro 55 Gin His Ala Thr Ile Pro Leu Leu Pro Asp Glu Ile Leu Ala Lys Cys 70 75 Gin Ala Arg Asn Ile Pro Ser Leu Thr Tyr Asn Gin Leu Ala Val Ile 90 Tyr Lys Leu Ile Trp Tyr Gin Asp Gly Tyr Glu Gln Pro Ser Glu Glu 100 105 110 Asp Leu Arg Arg Ile Met Ser Gin Pro Asp Glu Asn Glu Ser Gin Thr 115 120 125 WO 97/46079 PCT/US97/09682 Asp Leu 145 Gin Met Phe Met Ser 225 Ile Ile His Ile Phe 305 Ile Ile Ala Ser Pro 385 Thr Leu Ile Ser Val 130 Ile 3lu Leu Ala Ala 210 Met Phe Gin Cys Leu 290 ser Trp Thr Ser Thr 370 Gln Gln Gin Gin Val 450 Ser Val Asp Arg Asn 195 Asp Lys Ser Ser Gly 275 Thr Leu Asp Gin Val 355 ser Pro Pro Pro Pro 435 Thr Phe Glu Gin Met 180 Asn Asn Val Asp Tyr 260 Asp Glu Lys Val Glu 340 Gly Ala Gln Gin Gin 420 SGin Ala Arg Phe Ile 165 Ala Arg Ile Asp Arg 245 Tyr Ser Leu Leu His 325 Glu Gly Ala Pro Leu 405 Leu Pro Pro His Ala 150 Thr Arg Ser Glu Asn 230 Pro Ile Met Arg Lys 310 Ala Asn Ala Ala Ser 390 Gin Gin SGin Gly Ile 135 Lys Leu Arg Tyr Asp 215 Val Gly Asp Ser Thr 295 Asn Ile Glu Ile Ala 375 Ser Pro Pro Leu Ser 455 Thr Gly Leu Tyr Thr 200 Leu Glu Leu Thr Leu 280 Leu Arg Pro Arg Thr 360 Ala Leu Gin SGln Leu 440 Leu Glu Leu Lys Asp 185 Arg Leu Tyr Glu Leu 265 Val Gly Lys Pro Leu 345 Ala Ala Thr Leu Leu 425 Pro Ser Ile Pro Ala 170 His Asp His Ala Lys 250 Arg Phe Asn Leu Ser 330 Glu Gly Gln Gin Pro 410 Gin Val Ala 126 Thr Ala 155 Cys Ser Ser Phe Leu 235 Ala Ile Tyr Gln Pro 315 Val Arg Ile His Asn 395 SPro Thr Ser Val Ile 140 Phe Ser Ser Tyr Cys 220 Leu Gin Tyr Ala Asn 300 Lys Gin Ala Asp Gin 380 Asp Gin Gin Ala Ser 460 Leu Thr Ser Asp Lys 205 Arg Thr Leu Ile Lys 285 Ala Phe Ser Glu cys 365 Pro Ser Leu Leu Pro 445 Thr ,Thr Lys Glu Ser 190 Met Gn Ala Val Leu 270 Leu Glu Leu His Arg 350 Asp Gin Gln SGln SGln 430 SVal SSer val Ile Val 175 Ile Ala Met Ile Glu 255 Asn Leu Met Glu Leu 335 Met Ser Pro His Gly 415 Pro Pro Ser Gln Pro 160 Met Phe Gly Phe Val 240 Ala Arg Ser Cys Glu 320 Gln Arg Ala Gin Gln 400 Gln Gin Ala Glu Tyr 465 Met Gly Gly Ser Ala Ala Ile Gly Pro Ile Thr Pro Ala Thr Thr 470 475 480 WO 97/46079 PCT/US97/09682 127 Ser Ser Ile Thr Ala Ala Val Thr Ala Ser ser Thr Thr Ser Ala val 485 490 495 Pro Met Gly Asn Gly Val Gly Val Gly Val Gly Val Gly Gly Asn Val 500 505 510 Ser Met Tyr Ala Asn Ala Gin Thr Ala Met Ala Leu Met Gly Val Ala 515 520 525 Leu His Ser His Gin Glu Gin Leu Ile Gly Gly Val Ala Val Lys Ser 530 535 540 Glu His Ser Thr Thr Ala 545 550 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 855 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..853 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1..855 OTHER INFORMATION: /note= "domain glucocorticoid receptor ligand binding domain." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: ACA AAG AAA AAA ATC AAA GGG ATT CAG CAA GCC ACT GCA GGA GTC TCA 48 Thr Lys Lys Lys Ile Lys Gly Ile Gin Gin Ala Thr Ala Gly Val Ser 1 5 10 CAA GAC ACT TCG GAA AAT CCT AAC AAA ACA ATA GTT CCT GCA GCA TTA 96 Gin Asp Thr Ser Glu Asn Pro Asn Lys Thr Ile Val Pro Ala Ala Leu 25 CCA CAG CTC ACC CCT ACC TTG GTG TCA CTG CTG GAG GTG ATT GAA CCC 144 Pro Gin Leu Thr Pro Thr Leu Val Ser Leu Leu Glu Val Ile Glu Pro 40 GAG GTG TTG TAT GCA GGA TAT GAT AGC TCT GTT CCA GAT TCA GCA TGG 192 Glu Val Leu Tyr Ala Gly Tyr Asp Ser Ser Val Pro Asp Ser Ala Trp 55 AGA ATT ATG ACC ACA CTC AAC ATG TTA GGT GGG CGT CAA GTG ATT GCA 240 Arg Ile Met Thr Thr Leu Asn Met Leu Gly Gly Arg Gin Val Ile Ala 70 75 GCA GTG AAA TGG GCA AAG GCG ATA CTA GGC TTG AGA AAC TTA CAC CTC 288 Ala Val Lys Trp Ala Lys Ala Ile Leu Gly Leu Arg Asn Leu His Leu 90 GAT GAC CAA ATG ACC CTG CTA CAG TAC TCA TGG ATG TTT CTC ATG GCA 336 Asp Asp Gin Met Thr Leu Leu Gin Tyr Ser Trp Met Phe Leu Met Ala 100 105 110 WO 97/46079 WO 9746079PCTIUS97/09682 TTT GCC TTG GGT TGG AGA TCA Phe Ala Leu Gly Trp Arg Ser

TGC

cys

TGC

cys 145

CAA

Gin

CTG

Leu

TTT

Phe

GTC

Val

CTG

Leu 225

ACC

Thr

CCA

Pro

AAT

As n

TTT

Phe 130

ATG

Met

AGA

Arg

CTT

Leu

GAT

Asp

AAA

Ly s 210

ACA

Thr

TAC

Tyr

GAG

Giu

GGA

Gly

GCT

Ala

TAT

Tyr

TTG

Leu

CTC

Leu

GAG

Giu 195

AGG

Arg

AAG

Ly s

TGC

Cys

ATG

Met

AAT

Asn 275

CCT

Pro

GAC

Asp

CAG

Gin

TCC

Ser 180

ATT

Ile

GAA

GlU

CTT

Leu

TTC

Phe

TTA

Leu 260

ATC

Ile

GAT

Asp

CAA

Gin

GTA

Vai 165

TCA

Ser

CGA

Arg

GGG

Gly

CTG

Leu

CAG

Gin 245

GCT

Ala

AAA

Lys

CTG

Leu

TGT

Cy 5 150

TCC

Ser

GTT

Val

ATG

Met

AAC

As n

GAC

Asp 230

ACA

Thr

GAA

Giu

AAG

Lys

ATT

Ile 135

AAA

Lys

TAT

Tyr

GCT

Ala

ACT

Thr

TCC

Ser 215

TCC

Ser

TTT

Phe

ATC

Ile

CTT

Leu 128 TAC AGA CAA TCA Tyr Arg Gin ser 120 ATT AAT GAG CAG Ile Asn GiU Gin CAC ATG CTG TTT His Met Leu Phe 155 GAA GAG TAT CTC Giu Giu Tyr Leu 170 AAG GAA GGT CTG Lys Giu Gly Leu 185 TAT ATC AAA GAG Tyr Ile Lys Glu 200 AGT CAG AAC TGG Ser Gin Asn Trp ATG CAT GAG GTG Met His Giu Vai 235 TTG GAT AAG ACC Leu Asp Lys Thr 250 ATC ACT AAT CAG Ile Thr Asn Gin 265

AGC

Ser

AGA

Arg 140

GTC

Val

TGT

Cys

AAG

Ly s

CTA

Leu

CAA

Gin 220

GTT

Val

ATG

met

ATA

Ile

GGA

Giy 125

ATG

Met

TCC

Ser

ATG

met

AGC

ser

GGA

Gly 205

CGG

Arg

GAG

Giu

AGT

Ser

CCA

AAC

Asn

TCT

Ser

TCT

Ser

AAA

Lys

CAA

Gin 190

AAA

Lys

TTT

Phe

AAT

Asn

ATT

Ile

AAA

CTG

Leu

CTA

Leu

GAA

GiU

ACC

Thr 175

GAG

Giu

GCC

Ala

TAC

Tyr

CTC

Leu

GAA

GiU 255

TAT

CTC

Leu

CCC

Pro

TTA

Leu 160

TTA

Leu

TTA

Leu

ATC

Ile

CAA

Gin

CTT

Leu 240

TTC

Phe

TCA

672 Pro Lys Tyr Ser 270 CTG TTT CAT CAA AAA T GA Leu Phe His Gin Lys INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 284 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Thr Lys Lys Lys Ile Lys Giy Ile Gin Gin Ala Thr Ala Giy Val Ser 1 5 10 Gin Asp Thr Ser Giu Asn Pro Asn Lys Thr Ile Val Pro Ala Ala Leu 25 Pro Gin Leu Thr Pro Thr Leu Val Ser Leu Leu Giu Val Ile Glu Pro 40 Giu Val Leu Tyr Ala Giy Tyr Asp Ser ser Val Pro Asp Ser Ala Trp 55 WO 97/46079 WO 9746079PCTIUS97/09682 Arg Ala Asp Phe cy s Cys 145 Gin Leu Phe Val Leu 225 Thr Pro As n Ile Val Asp Ala Phe 130 Met Arg Leu Asp Lys 210 Thr Tyr GiU Gly met Lys Gin Leu 115 Ala Tyr Leu Leu Giu 195 Arg Lys Cys Met Asn 275 Thr Trp Met 100 Gly Pro Asp Gin ser 180 Ile Giu Leu Phe Leu 260 Ile Thr Ala Thr Trp Asp Gin Val 165 Ser Arg Gly Leu Gin 245 Ala Ly s Leu 70 Lys Leu Arg Leu Cy 5 150 ser Val Met As n Asp 230 Thr Giu As n Ala Leu ser Ile 135 Lys Tyr Ala Thr Ser 215 Ser Phe Ile Met Leu Ile Leu Gin Tyr 105 Tyr Arg 120 Ile Asn His met Giu Giu Lys Giu 185 Tyr Ile 200 Ser Gin Met His Leu Asp Ile Thr 265 Gly Gly 90 Ser Gin Giu Leu Tyr 170 Gly Ly s Asn Giu Lys 250 Asn His 129 Gly 75 Leu Trp Ser Gin Phe 155 Leu Leu Glu Trp Val 235 Thr Gin Gin Arg Arg met ser Arg 140 Val Cys Lys Leu Gin 220 Val Met Ile Lys Gin Asn Phe Gly 125 Met ser Met Ser Gly 205 Arg Giu Ser Pro Val Leu Leu 110 Asnf Ser Ser Lys Gin 190 Ly s Phe Asn Ile Lys 270 Ile His met Leu Leu Giu Thr 175 Giu Ala Tyr Leu Giu 255 Tyr Ala Leu Ala Leu Pro Leu 160 Leu Leu Ile Gin Leu 240 Phe Ser Lys Leu Leu Phe 280 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 50 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1.-50 OTHER INFORMATION: /note= "element copper inducible regulatory element (ACEl binding site).,, (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: AGCTTAGCGA TGCGTCTTTT CCGCTGAACC GTTCCAGCAA AAAAGACTAG WO 97/46079 PCT/US97/09682 130 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ix) FEATURE: NAME/KEY: misc_feature LOCATION: 1..19 OTHER INFORMATION: /note= "element tet operator." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: ACTCTATCAG TGATAGAGT 19 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1..29 OTHER INFORMATION: /note= "element ecdysone response element." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: GATCCGACAA GGGTTCAATG CACTTGTCA 29 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 371 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1..371 OTHER INFORMATION: /note= "element heat shock inducible regulatory element (HSP81-1 promoter)." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: GTGGAGTCTC GAAACGAAAA GAACTTTCTG GAATTCGTTT GCTCACAAAG CTAAAAACGG TTGATTTCAT CGAAATACGG CGTCGTTTTC AAAGAACAAT CCAGAAATCA CTGGTTTTCC 120 TTTATTTCAA AAGAAGAGAC TAGAACTTTA TTTCTCCTCT ATAAAATCAC TTTGTTTTTC 180 CCTCTCTTCT TCATAAATCA ACAAAACAAT CACAAATCTC TCGAAACGCT CTCGAAGTTC 240 CAAATTTTCT CTTAGCATTC TCTTTCGTTT CTCGTTTGCG TTGAATCAAA GTTCGTTGCG 300 WO 97/46079 PCT/US97/09682 131 ATGGCGGATG TTCAGATGGC TGATGCAGAG ACTTTTGCTT TCCAAGCTGA GATTAACCAG 360 CTTCTTAGCT T 371 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID GGATCCGGAT CAAAAATGGG AAGGGGTAG 29 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: GGATCCGCTG CGGCGAAGCA GCCAAGGTTG

Claims

1. A recombinant nucleic acid molecule, comprising an inducible regulatory element operably linked to a nucleic acid molecule encoding CAULIFLOWER (CAL).

2. The recombinant nucleic acid molecule of claim 1, wherein said inducible regulatory element is selected from the group consisting of a: copper inducible regulatory element; tetracycline inducible regulatory element; ecdysone inducible regulatory element; and heat-shock inducible regulatory element.

3. The recombinant nucleic acid molecule of claim 2, wherein said CAL has an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 12.

4. A transgenic seed plant, comprising the recombinant nucleic acid molecule of claim 1.

5. The transgenic seed plant of claim 4, wherein said seed plant is an angiosperm. 20

6. The transgenic seed plant of claim 4, wherein said seed plant is a gymnosperm.

7. A method of converting shoot meristem to S. floral meristem in an angiosperm, comprising the steps of: introducing into said angiosperm a o 25 recombinant nucleic acid molecule comprising an inducible regulatory element operably linked to a nucleic acid molecule encoding CAULIFLOWER (CAL) to produce a transgenic angiosperm; and contacting said transgenic angiosperm S 30 with an inducing agent, thereby increasing expression of said CAL and converting shoot meristem to floral meristem in said transgenic angiosperm.

8. The method of claim 7, wherein said inducible regulatory element is selected from the group consisting of a: copper inducible regulatory element; tetracycline inducible regulatory element; \BIS \homeS\Isabe lfI\Spei\ 3296 .doc 19/07/00 133 Secdysone inducible regulatory element; and Sheat-shock inducible regulatory element.

9. A method of promoting early reproductive development in a seed plant,-comprising the steps of: introducing into said seed plant said recombinant nucleic acid molecule comprising an inducible regulatory element operably linked to a nucleic acid molecule encoding CAULIFLOWER (CAL) to produce a transgenic seed plant; and contacting said transgenic seed plant with an inducing agent, thereby increasing expression of said CAL and promoting early reproductive development in said transgenic seed plant. The method of claim 9, wherein said inducible regulatory element is selected from the group consisting of a: copper inducible regulatory element; tetracycline inducible regulatory element; ecdysone inducible regulatory element; and 20 heat-shock inducible regulatory element.

11. A nucleic acid molecule encoding a chimeric protein, comprising a nucleic acid molecule encoding a S. floral meristem identity gene product linked in frame to a nucleic acid molecule encoding a ligand binding domain.

12. The nucleic acid molecule of claim 11, S: wherein said floral meristem identity gene product is selected from the group consisting of AP1, CAL and LFY.

13. The nucleic acid molecule of claim 11, Swherein said ligand binding domain is a steroid binding 30 domain.

14. The nucleic acid molecule of claim 13, wherein said steroid binding domain is selected from the group consisting of an ecdysone receptor ligand binding domain and a glucocorticoid receptor ligand binding domain. The nucleic acid molecule of claim 14, 7- wherein said floral meristem identity gene product is AP1. BRISI\IOOr\r.Sabe1H\Speci\32 9 67 .doc 19/07/00 134

16. The nucleic acid molecule of claim wherein said API has an amino acid sequence selected from Sthe group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8.

17. The nucleic acid molecule of claim 14, wherein said floral meristem identity gene product is CAL.

18. The nucleic acid molecule of claim 17, wherein said CAL has an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 12.

19. The nucleic acid molecule of claim 14, wherein said floral meristem identity gene product is LFY. The nucleic acid molecule of claim 19, wherein said LFY has the amino acid sequence of SEQ ID NO: 16.

21. A transgenic seed plant, comprising the nucleic acid molecule of claim 11.

22. The transgenic seed plant of claim 21, wherein said transgenic seed plant is an angiosperm. 20 23. The transgenic seed plant of claim 21, 20 wherein said transgenic seed plant is an gymnosperm.

24. The transgenic seed plant of claim 21, S* wherein said floral meristem identity gene product is Sselected from the group consisting of API, CAL and LFY. The transgenic seed plant of claim 24, e* 25 wherein said floral meristem identity gene product is API.

26. The transgenic seed plant of claim 24, wherein said floral meristem identity gene product is CAL.

27. The transgenic seed plant of claim 24, wherein said floral meristem identity gene product is LFY. 30 28. A method of converting shoot meristem to floral meristem in an angiosperm, comprising the steps of: introducing into said angiosperm said nucleic acid molecule of claim 26 to produce a transgenic angiosperm, wherein, under appropriate conditions, said chimeric protein containing a floral meristem identity gene product fused to a ligand binding domain is S'N expressed; and \\BpISl\home IsabelH\Specl i2967 doc 19/07/00 135 contacting said transgenic angiosperm (with cognate ligand, wherein, upon binding of said cognate ligand to said ligand binding domain, floral meristem identity gene product activity is increased, thereby converting shoot meristem to floral meristem in said transgenic angiosperm.

29. The method of claim 28, wherein said ligand binding domain is selected from the group consisting of an ecdysone receptor ligand binding domain and a glucocorticoid receptor ligand binding domain and said floral meristem identity gene product is AP1. The method of claim 28, wherein said ligand binding domain is selected from the group consisting of an ecdysone receptor ligand binding domain and a glucocorticoid receptor ligand binding domain and said floral meristem identity gene product is CAL.

31. The method of claim 28, wherein said ligand binding domain is selected from the group consisting of an ecdysone receptor ligand binding domain and a glucocorticoid receptor ligand binding domain and said floral meristem identity gene product is LFY.

32. A method of promoting early reproductive .development in a seed plant, comprising the steps of: introducing into said seed plant said 25 nucleic acid molecule of claim 26 to produce a transgenic seed plant, wherein, under appropriate conditions, said 5chimeric protein containing a floral meristem identity 0000 gene product fused to a ligand binding domain is expressed; and 30 contacting said transgenic seed plant with cognate ligand, wherein, upon binding of said cognate ligand to said ligand binding domain, floral meristem identity gene product activity is increased, thereby promoting early reproductive development in said transgenic seed plant.

33. The method of claim 32, wherein said ligand N binding domain is selected from the group consisting of an \\BPISL\home$\ Iab1H\Spci\3 2967 .doc 19/07/00 136 ecdysone receptor ligand binding domain and a Sglucocorticoid receptor ligand binding domain and said r floral meristem identity gene product is AP1. S34. The method of-claim 32, wherein said ligand binding domain is selected from the group consisting of an ecdysone receptor ligand binding domain and a glucocorticoid receptor ligand binding domain and said floral meristem identity gene product is CAL. The method of claim 32, wherein said ligand binding domain is selected from the group consisting of an ecdysone receptor ligand binding domain and a glucocorticoid receptor ligand binding domain and said floral meristem identity gene product is LFY.

36. The method of claim 7, wherein said CAL has an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 12.

37. The method of claim 9, wherein said CAL has an amino acid sequence selected from the group consisting of SEQ ID NO; 10 and SEQ ID NO: 12. 0000 00 00 8 0000 o 0 0 0 0 0000 0 0000 o 0 0 00* *000 0 0000 0 0 \\BP.ISI'ho.,5\IsablH\Speci\329r7.1doc 19/07100